Compounds having β2 adrenergic receptor agonist and muscarinic receptor antagonist activity

ABSTRACT

The invention is directed to compounds of formula I: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7a , R 7b , W, G 1 , G 2 , a, b, c, d and m are as defined in the specification, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. The invention is also directed to pharmaceutical compositions comprising such compounds; methods of using such compounds; and process and intermediates for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/992,927, filed Nov. 19, 2004 now U.S. Pat. No. 7,345,060; whichapplication claims the benefit of U.S. Provisional Application No.60/524,234, filed on Nov. 21, 2003; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds having β₂ adrenergicreceptor agonist and a muscarinic receptor antagonist activity. Thisinvention also relates to pharmaceutical compositions comprising suchcompounds, processes and intermediates for preparing such compounds andmethods of using such compounds to treat pulmonary disorders.

2. State of the Art

Pulmonary disorders, such as asthma and chronic obstructive pulmonarydisease (COPD), are commonly treated with bronchodilators. One class ofbronchodilator in widespread use consists of β₂ adrenergic receptor(adrenoceptor) agonists, such as albuterol, formoterol and salmeterol.These compounds are generally administered by inhalation. Another classof bronchodilator consists of muscarinic receptor antagonists(anticholinergic compounds), such as ipratropium and tiotropium. Thesecompounds are also typically administered by inhalation.

Pharmaceutical compositions containing both a β₂ adrenergic receptoragonist and a muscarinic receptor antagonist are also known in the artfor use in treating pulmonary disorders. For example, U.S. Pat. No.6,433,027 discloses medicament compositions containing a muscarinicreceptor antagonist, such as tiotropium bromide, and a β₂ adrenergicreceptor agonist, such as formoterol fumarate.

Although compositions containing both a β₂ adrenergic receptor agonistand a muscarinic receptor antagonist are known, it would be highlydesirable to provide compounds having both β₂ adrenergic receptoragonist and muscarinic receptor antagonist activity in the samemolecule. Compounds possessing both β₂ adrenergic receptor agonist andmuscarinic receptor antagonist activity are expected to be particularlyuseful as therapeutic agents since such bifunctional compounds wouldprovide bronchodilation through two independent modes of action whilehaving single molecule pharmacokinetics.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that have been found topossess both β₂ adrenergic receptor agonist and muscarinic receptorantagonist activity. Such compounds are expected to be useful astherapeutic agents for treating pulmonary disorders. Certain compoundsof this invention have also been found to possess affinity for dopamineD₂ receptors.

Accordingly, in one of its composition aspects, the present invention isdirected to a compound of formula I:

wherein

one of G¹ and G² represents NH and the other represents S, NH, O or CH₂;

W represents O or NW^(a); where W^(a) is hydrogen or (1-4C)alkyl;

each R¹ is independently selected from (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(1a), —C(O)OR^(1b),—SR^(1c), —S(O)R^(1d), —S(O)₂R^(1e) and —NR^(1f)R^(1g); where each ofR^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f) and R^(1g) isindependently hydrogen, (1-4C)alkyl or phenyl-(1-4C)alkyl;

each R² is independently selected from (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(2a), —C(O)OR^(2b),—SR^(2c), —S(O)R^(2d), S(O)₂R^(2e) and —NR^(2f)R^(2g); where each ofR^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f) and R^(2g) isindependently hydrogen, (1-4C)alkyl or phenyl-(1-4C)alkyl;

each R³ is independently selected from (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(3a), —C(O)OR^(3b),—SR^(3c), —S(O)R^(3d), —S(O)₂R^(3e) and —NR^(3f)R^(3g); or two R³ groupsare joined to form (1-3C)alkylene, (2-3C)alkenylene or oxiran-2,3-diyl;where each of R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f) and R^(3g)is independently hydrogen or (1-4C)alkyl;

R⁴ represents a divalent hydrocarbon group containing from 4 to 28carbon atoms and optionally containing from 1 to 10 heteroatoms selectedindependently from halo, oxygen, nitrogen and sulfur, provided that thenumber of contiguous atoms in the shortest chain between the twonitrogen atoms to which R⁴ is attached is in the range of from 4 to 16;

R⁵ represents hydrogen or (1-4C)alkyl;

R⁶ represents hydrogen or hydroxyl;

each R^(7a) and R^(7b) is independently selected from hydrogen,(1-4C)alkyl, hydroxy and fluoro;

a is 0 or an integer of from 1 to 3;

b is 0 or an integer of from 1 to 3;

c is 0 or an integer of from 1 to 4;

d in 0 or an integer of from 1 to 5; and

m is 0 or an integer of from 1 to 3;

or a pharmaceutically acceptable salt or solvate or stereoisomerthereof.

Among the compounds of formula I, compounds of particular interest arethose having an inhibitory constant (K_(i)) for the M₃ muscarinicreceptor less than about 100 nM and a half maximal effectiveconcentration EC₅₀ for agonism at the β₂ adrenergic receptor of lessthan about 100 nM. In particular, compounds of special interest arethose in which the ratio of the inhibitory constant (K_(i)) for the M₃muscarinic receptor to the EC₅₀ for agonism of the β₂ adrenergicreceptor ranges from about 30:1 to about 1:30.

In another of its composition aspects, this invention is directed to acompound of formula Ia:

wherein

R¹, R², R³, R⁴, R⁵, R⁶, G¹, G², W, a, b and c is as defined herein(including any specific or preferred embodiments);

or a pharmaceutically acceptable salt or solvate or stereoisomerthereof.

In another of its composition aspects, this invention is directed to acompound of formula Ib:

wherein

R¹, R², R³, R⁴, W, a, b and c is as defined herein (including anyspecific or preferred embodiments);

or a pharmaceutically acceptable salt or solvate or stereoisomerthereof.

In yet another of its composition aspects, this invention is directed toa compound of formula Ic:

wherein

R¹, R², R³, R⁴, W, a, b and c is as defined herein (including anyspecific or preferred embodiments);

or a pharmaceutically acceptable salt or solvate or stereoisomerthereof.

In still another of its composition aspects, this invention is directedto a compound of formula Id:

wherein

R⁴ is as defined herein (including any specific or preferredembodiments);

or a pharmaceutically acceptable salt or solvate or stereoisomerthereof.

In another of its composition aspects, this invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of thisinvention or a pharmaceutically acceptable salt or solvate orstereoisomer thereof. Such pharmaceutical compositions may optionallycontain other therapeutic agents.

Accordingly, in one embodiment, this invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of thisinvention or a pharmaceutically acceptable salt or solvate orstereoisomer thereof; and a therapeutically effective amount of asteroidal anti-inflammatory agent, such as a corticosteroid, or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

In another embodiment, this invention is directed to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of a compound of this invention or apharmaceutically acceptable salt or solvate or stereoisomer thereof; anda therapeutically effective amount of a phosphodiesterase-4 (PDE4)inhibitor or a pharmaceutically acceptable salt or solvate orstereoisomer thereof.

Compounds of this invention possess both β₂ adrenergic receptor agonistactivity and muscarinic receptor antagonist activity. Accordingly, thecompounds of this invention are expected to be useful for treatingpulmonary disorders, such as asthma and chronic obstructive pulmonarydisease.

Accordingly, in one of its method aspects, this invention is directed toa method for treating a pulmonary disorder, the method comprisingadministering to a patient in need of treatment a therapeuticallyeffective amount of a compound of this invention or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof.

Additionally, in another of its method aspects, this invention isdirected to a method of producing bronchodilation in a patient, themethod comprising administering to the patient abronchodilation-producing amount of a compound of this invention or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

This invention is also directed to a method of treating chronicobstructive pulmonary disease or asthma, the method comprisingadministering to a patient in need of treatment a therapeuticallyeffective amount of a compound of this invention or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof.

This invention is also directed to processes and novel intermediatesuseful for preparing compounds of formula I or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof. Accordingly, inanother of its method aspects, this invention is directed to a processof preparing a compound of formula I, the process comprising:

(a) reacting a compound of formula 1 or a salt thereof, with a compoundof formula 2;

(b) reacting a compound of formula 3 or a salt thereof, with a compoundof formula 4;

(c) coupling a compound of formula 5 with a compound of formula 6;

(d) for a compound of formula I wherein R⁵ represents a hydrogen atom,reacting a compound of formula 3 with a compound of formula 7a or 7b ora hydrate thereof, in the presence of a reducing agent;

(e) reacting a compound of formula 1 with a compound of formula 8 or ahydrate thereof, in the presence of a reducing agent;

(f) reacting a compound of formula 9, with a compound of formula 10; or

(g) reacting a compound of formula 11 or a hydrate thereof, with acompound of formula 10, in the presence of a reducing agent;

and then removing any protecting groups to form a compound of formula I;wherein the compounds of formula 1-11 are as defined therein.

In one embodiment, the above process further comprises the step offorming a pharmaceutically acceptable salt of a compound of formula I.In other embodiments, this invention is directed to the other processesdescribed herein; and to the product prepared by any of the processesdescribed herein.

This invention is also directed to a compound of this invention or apharmaceutically acceptable salt or solvate or stereoisomer thereof, foruse in therapy or as a medicament.

Additionally, this invention is directed to the use of a compound ofthis invention or a pharmaceutically acceptable salt or solvate orstereoisomer thereof, for the manufacture of a medicament; especiallyfor the manufacture of a medicament for the treatment of a pulmonarydisorder.

DETAILED DESCRIPTION OF THE INVENTION

In one of its composition aspects, this invention is directed to novelcompounds of formula I or pharmaceutically acceptable salts or solvatesor stereoisomers thereof. These compounds may contain one or more chiralcenters and therefore, when such chiral centers are present, thisinvention is directed to racemic mixtures; pure stereoisomers (i.e.,enantiomers or diastereomers); stereoisomer-enriched mixtures and thelike unless otherwise indicated. When a particular stereoisomer is shownor named herein, it will be understood by those skilled in the art thatminor amounts of other stereoisomers may be present in the compositionsof this invention unless otherwise indicated, provided that the utilityof the composition as a whole is not eliminated by the presence of suchother isomers.

In particular, compounds of formula I in which R⁶ represents a hydroxylgroup contain a chiral center at the carbon atom indicated by thesymbol * in the following formula:

In one embodiment of this invention, the carbon atom identified by thesymbol * has the (R) configuration. In this embodiment, it is preferredfor compounds of formula I to have the (R) configuration at the carbonatom identified by the symbol * or to be enriched in a stereoisomericform having the (R) configuration at this carbon atom. In anotherembodiment of this invention, the carbon atom identified by the symbol *has the (S) configuration. In this embodiment, it is preferred forcompounds of formula I to have the (S) configuration at the carbon atomidentified by the symbol * or to be enriched in a stereoisomeric formhaving the (S) configuration at this carbon atom. In some cases, inorder to optimize the β₂ adrenergic agonist activity of the compounds ofthis invention, it is preferred that the carbon atom identified by thesymbol * has the (R) configuration.

The compounds of this invention also contain several basic groups (e.g.,amino groups) and therefore, such compounds can exist as the free baseor in various salt forms. All such salt forms are included within thescope of this invention. Furthermore, solvates of compounds of thisinvention or salts thereof are included within the scope of thisinvention.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds ofthis invention are included within the scope of this invention unlessotherwise specified.

The nomenclature used herein to name the compounds of this invention andintermediates thereof has generally been derived using thecommercially-available AutoNom software (MDL, San Leandro, Calif.). Forexample, compounds of formula I wherein W is O have typically been namedas ester derivatives of biphenyl-2-ylcarbamic acid; and compounds offormula I wherein W is NW^(a) have been named as urea derivatives.

REPRESENTATIVE EMBODIMENTS

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of thisinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of this invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from this invention unless specifically indicated.

In particular embodiments of the compounds of formula I, a and b areindependently 0, 1 or 2; including 0 or 1. In one embodiment, both a andb are both 0.

When present, each R¹ may be at the 2, 3, 4, 5 or 6-position of thephenyl ring to which it is attached. In one embodiment, each R¹ isindependently selected from (1-4C)alkyl, halo, —OR^(1a) and—NR^(1f)R^(1g); such as methyl, fluoro, chloro, bromo, hydroxy, methoxy,amino, methylamino, dimethylamino and the like. Particular values for R¹are fluoro or chloro.

When present, each R² may be at the 3, 4, 5 or 6-position on thephenylene ring to which it is attached (where the carbon atom on thephenylene ring attached to the nitrogen atom is position 1). In oneembodiment, each R² is independently selected from (1-4C)alkyl, halo,—OR^(2a) and —NR^(2f)R^(2g); such as methyl, fluoro, chloro, bromo,hydroxy, methoxy, amino, methylamino, dimethylamino and the like.Particular values for R² are fluoro or chloro.

Each R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f) and R^(1g) andR^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f) and R^(2g) as used in R¹and R², respectively, is independently hydrogen, (1-4C)alkyl orphenyl-(1-4C)alkyl; such as hydrogen, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl and benzyl. In oneembodiment, these groups are independently hydrogen or (1-3C)alkyl. Inanother embodiment, these groups are independently hydrogen, methyl orethyl.

In one embodiment of this invention, W is O. In another embodiment, W isNW^(a).

Generally, it has been found that compounds in which W represents Oexhibit particularly high affinity for muscarinic and β₂ adrenergicreceptors. Accordingly, in a particular embodiment of this invention, Wpreferably represents O.

When W is NW^(a), W^(a) is hydrogen or (1-4C)alkyl; such as hydrogen,methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl andtert-butyl. In one embodiment, W^(a) is hydrogen or (1-3C)alkyl. Inanother embodiment, W^(a) is hydrogen, methyl or ethyl; such as hydrogenor methyl. In yet another embodiment, W^(a) is hydrogen and NW^(a) isNH.

When b is 0 and m is 2 (such that W is attached to a piperidine ring), aparticular embodiment of interest are compounds wherein W is attached tothe piperidine ring at the 4-position with respect to the nitrogen atomof the piperidine ring.

In a particular embodiment of the compounds of formula I, c is 0, 1 or2; including 0 or 1. In one embodiment, c is 0.

In a particular embodiment of the compounds of formula I, d is 0, 1 or2; including 0 or 1. In one embodiment, d is 0. In another embodiment, dis 1.

In one embodiment, R^(7a) and R^(7b) are independently selected fromhydrogen, methyl or ethyl. A particular value for R^(7a) and R^(7b) ishydrogen.

In a particular embodiment of the compounds of formula I, m is 0, 1 or2; including 1 or 2. In one embodiment, m is 2. When m is 1, theresulting ring is a pyrrolidine ring; and when m is 2, the resultingring is a piperidine ring.

In a particular embodiment of the compounds of formula I, c is 0, 1 or2; including 0 or 1. In one embodiment, c is 0.

In a particular embodiment, each R³ is independently selected from(1-4C)alkyl; such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl and tert-butyl. In another aspect, each R³ isindependently methyl or ethyl.

When m is 2, in one embodiment, each R³ is at the 3, 4 or 5-position onthe piperidine ring (where the nitrogen atom of the piperidine ring isposition 1). In another embodiment, R³ is at 4-position on thepiperidine ring. In yet another embodiment, each R³ is at the 2 or6-position of the piperidine ring.

In another embodiment, R³ is at the 1-position of the piperidine ring,i.e., on the nitrogen atom of the piperidine ring thus forming aquaternary amine salt. In a particular aspect of this embodiment, eachR³ is independently selected from (1-4C)alkyl; such as methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl. Inanother aspect, each R³ is independently methyl or ethyl.

In yet another embodiment, two R³ groups are joined to form a(1-3C)alkylene or (2-3C)alkenylene group. For example, two R³ groups atthe 2 and 6-positions on the piperidine ring can be joined to form anethylene bridge (i.e., the piperidine ring and the R³ groups form an8-azabicyclo[3.2.1]octane ring); or two R³ groups at the 1 and4-positions on the piperidine ring can be joined to form an ethylenebridge (i.e., the piperidine ring and the R³ groups form an1-azabicyclo[2.2.2]octane ring); or two R³ groups at the 2 and6-positions on the piperidine ring can be joined to form an ethenylenebridge (i.e., the piperidine ring and the R³ groups form an8-azabicyclo[3.2.1]oct-6-ene ring). In this embodiment, other R³ groupsas defined herein may also be present.

In still another embodiment, two R³ groups are joined to form aoxiran-2,3-diyl group. For example, two R³ groups at the 2 and6-positions on the piperidine ring can be joined to form a3-oxatricyclo[3.3.1.0^(2,4)]nonane ring). In this embodiment, other R³groups as defined herein may also be present.

Each R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f) and R^(3g) as usedin R³ is independently hydrogen or (1-4C)alkyl; such as hydrogen,methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl andtert-butyl. In one embodiment, these groups are independently hydrogenor (1-3C)alkyl. In another embodiment, these groups are independentlyhydrogen, methyl or ethyl.

In one embodiment of the compounds of formula I, R⁵ is hydrogen or(1-4C)alkyl; such as hydrogen, methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, isobutyl and tert-butyl. In another embodiment, eachR⁵ is independently hydrogen, methyl or ethyl. In a particularembodiment, R⁵ is hydrogen.

In one embodiment of this invention, R⁶ is hydrogen. In anotherembodiment, R⁶ is hydroxyl.

In separate embodiments, G¹ and G² are selected from:

G¹ is NH and G² is S;

G¹ is NH and G² is NH;

G¹ is NH and G² is O;

G¹ is NH and G² is CH₂;

G¹ is S and G² is NH;

G¹ is O and G² is NH; and

G¹ is CH₂ and G² is NH.

The divalent hydrocarbon group of this invention, R⁴, contains from 4 to28 carbon atoms and optionally contains from 1 to 10 heteroatomsselected independently from halo, oxygen, nitrogen and sulfur, providedthat the number of contiguous atoms in the shortest chain between thetwo nitrogen atoms to which R⁴ is attached is in the range of from 4 to16. In one embodiment, this group contains from 4 to 24 carbon atoms,including from 6 to 20 carbon atoms, such as from 8 to 18 carbon atoms;and optionally contains from 1 to 8 heteroatoms, including from 1 to 6heteroatoms.

The divalent hydrocarbon may contain any arrangement of atoms includingalkylene, cycloalkylene, arylene, heteroarylene and heterocyclene groupsor combinations thereof. The hydrocarbon group may be interrupted by oneor more heteroatoms or combinations of heteroatoms and carbon atoms toform various functional groups, such as ethers, thioethers, amines,amides, esters, carbamates, ureas, sulfones, sulfoxides, sulfonamidesand the like.

When determining the number of contiguous atoms in the shortest chainbetween the two nitrogen atoms to which divalent hydrocarbon group isattached, each contiguous atom of the chain is counted consecutivelystarting from the first atom in the divalent hydrocarbon group, i.e.,the atom adjacent to the nitrogen atom of the azacycloalkyl group (i.e.,a piperidinyl group when m is 2) in formula I, and ending with the lastatom in the divalent hydrocarbon group, i.e., the atom adjacent tonitrogen atom the —NHCH(R⁵)— group in formula I. Where two or morechains are possible, the shortest chain is used to determine the numberof contiguous atoms. As shown below, for example, when the divalenthydrocarbon group is —(CH₂)₂—NHC(O)—CH₂-(phen-1,4-ylene)-CH₂—, there are10 contiguous atoms in the shortest chain as shown below:

In a particular aspect of this invention, the divalent hydrocarbon groupof the compounds of this invention (e.g., R⁴ in formula I) is a divalentgroup of the formula:—(R^(4a))_(d)-(A¹)_(e)-(R^(4b))_(f)-Q-(R^(4c))_(g)-(A²)_(h)-(R^(4d))_(i)—wherein

d, e, f, g, h and i are each independently selected from 0 and 1;

R^(4a), R^(4b), R^(4c) and R^(4d) are each independently selected from(1-10C)alkylene, (2-10C)alkenylene and (2-10C)alkynylene, wherein eachalkylene, alkenylene or alkynylene group is unsubstituted or substitutedwith from 1 to 5 substituents independently selected from (1-4C)alkyl,fluoro, hydroxy, phenyl and phenyl-(1-4C)alkyl;

A¹ and A² are each independently selected from (3-7C)cycloalkylene,(6-10C)arylene, —O-(6-10C)arylene, (6-10C)arylene-O—,(2-9C)heteroarylene, —O-(2-9C)heteroarylene, (2-9C)heteroarylene-O— and(3-6C)heterocyclene, wherein each cycloalkylene is unsubstituted orsubstituted with from 1 to 4 substitutents selected independently from(1-4C)alkyl, and each arylene, heteroarylene or heterocyclene group isunsubstituted or substituted with from 1 to 4 substituents independentlyselected from halo, (1-4C)alkyl, (1-4C)alkoxy, —S-(1-4C)alkyl,—S(O)-(1-4C)alkyl, —S(O)₂-(1-4C)alkyl, —C(O)O(1-4C)alkyl, carboxy,cyano, hydroxy, nitro, trifluoromethyl and trifluoromethoxy;

Q is selected from a bond, —O—, —C(O)O—, —OC(O)—, —S—, —S(O)—, —S(O)₂—,—N(Q^(a))C(O)—, —C(O)N(Q^(b))-, —N(Q^(c))S(O)₂—, —S(O)₂N(Q^(d))-,—N(Q^(e))C(O)N(Q^(f))-, —N(Q^(g))S(O)₂N(Q^(h))-, —OC(O)N(Q^(i))-,—N(Q^(j))C(O)O— and —N(Q^(k));

Q^(a), Q^(b), Q^(c), Q^(d), Q^(e), Q^(f), Q^(g), Q^(h), Q^(i), Q^(j) andQ^(k) are each independently selected from hydrogen, (1-6C)alkyl, A³ and(1-4C)alkylene-A⁴, wherein the alkyl group is unsubstituted orsubstituted with from 1 to 3 substituents independently selected fromfluoro, hydroxy and (1-4C)alkoxy; or together with the nitrogen atom andthe group R^(4b) or R^(4c) to which they are attached, form a 4-6membered azacycloalkylene group;

A³ and A⁴ are each independently selected from (3-6C)cycloalkyl,(6-10C)aryl, (2-9C)heteroaryl and (3-6C)heterocyclyl, wherein eachcycloalkyl is unsubstituted or substituted with from 1 to 4substitutents selected independently from (1-4C)alkyl and each aryl,heteroaryl or heterocyclyl group is unsubstituted or substituted withfrom 1 to 4 substituents independently selected from halo, (1-4C)alkyland (1-4C)alkoxy.

In this embodiment, the values of each of the components R^(4a), A¹,R^(4b), Q, R^(4c), A² and R^(4d) are selected such that the number ofcontiguous atoms in the shortest chain between the two nitrogen atoms towhich divalent hydrocarbon group is attached is in the range of from 4to 16, (specifically, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16);including 8, 9, 10, 11, 12, 13 or 14; such as 8, 9, 10 or 11; or 9 or10. When selecting values for each variable, it will be appreciated bythose skilled in the art that values should be selected such that achemically stable group is formed.

In one embodiment, R^(4a) is selected from (1-10C)alkylene,(2-10C)alkenylene and (2-10C)alkynylene wherein the alkylene group isunsubstituted or substituted with 1 or 2 substituents independentlyselected from (1-4C)alkyl, hydroxy and phenyl. Representative examplesof particular values for R^(4a) are —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—,—(CH₂)₅—, —(CH₂)₆—, —(CH₂)₇—, —(CH₂)₈—, —(CH₂)₉—, —(CH₂)₁₀—,—(CH₂)CH(CH₃)—, —(CH₂)C(CH₃)₂—, and —(CH₂)₂C(phenyl)₂—. In anotheraspect, R^(4a) is —(CH₂)C(═CH₂)—.

In one embodiment, d is 1.

In one embodiment, A¹ is an optionally substituted (3-7C)cycloalkylenegroup; including a cyclohexylene group, such as cyclohex-1,4-ylene andcyclohex-1,3-ylene; and a cyclopentylene group, such ascyclopent-1,3-ylene.

In another embodiment, A¹ is an optionally substituted (6-10C)arylenegroup, including a phenylene group, such as phen-1,4-ylene,phen-1,3-ylene and phen-1,2-ylene; and a naphthylene group, such asnaphth-1,4-ylene and napth-1,5-ylene.

In yet another embodiment, A¹ is an optionally substituted(2-9C)heteroarylene group, including a pyridylene group, such aspyrid-1,4-ylene; a furylene group, such as fur-2,5-ylene andfur-2,4-ylene; a thienylene group, such as thien-2,5-ylene andthien-2,4-ylene; and a pyrrolylene, such as pyrrol-2,5-ylene andpyrrol-2,4-ylene.

In still another embodiment, A¹ is an optionally substituted(3-6C)heterocyclene group, including a piperidinylene group, such aspiperidin-1,4-ylene; and a pyrrolidinylene group, such aspyrrolidin-2,5-ylene.

In a particular embodiment, A¹ is an optionally substituted phenylene,thienylene, cyclopentylene, cyclohexylene or piperidinylene.

In one embodiment, e is 0.

In a particular embodiment, R^(4b) is (1-5C)alkylene. Representativeexamples of particular values for R^(4b) are —CH₂—, —(CH₂)₂—, —(CH₂)₃—,—(CH₂)₄—, —(CH₂)₅—; including methylene, ethylene and propylene.

In one embodiment, f is 0.

In a particular embodiment, Q is selected from a bond, —N(Q^(a))C(O)—,—C(O)N(Q^(b))-, —N(Q^(c))S(O)₂—, —S(O)₂N(Q^(d))-,—N(Q^(e))C(O)N(Q^(f))-, —OC(O)N(Q^(i))-, —N(Q^(j))C(O)O— or —N(Q^(k));such as where Q is a bond, —N(Q^(a))C(O)— or —C(O)N(Q^(b))-.Representative examples of particular values for Q are a bond, O, NH,—C(O)NH—, —C(O)N(CH₃)—, —NHC(O)—, —N(CH₃)C(O)—, —S(O)₂NH—,—S(O)₂N(CH₃)—, —NHS(O)₂—, —N(CH₃)S(O)₂— and —NHC(O)NH—. Another exampleof a value for Q, together with R^(4c), is —C(O)(piperidin-1,4-ylene).

In one embodiment, Q^(a), Q^(b), Q^(c), Q^(d), Q^(e), Q^(f), Q^(g),Q^(h), Q^(i), Q^(j) and Q^(k) are each independently selected fromhydrogen and (1-6C)alkyl, wherein the alkyl group is unsubstituted orsubstituted with from 1 to 3 substituents independently selected fromfluoro, hydroxy and (1-4C)alkoxy. For example, Q^(a), Q^(b), Q^(c),Q^(d), Q^(e), Q^(f), Q^(g), Q^(h), Q^(i), Q^(j) and Q^(k) are eachindependently selected from hydrogen, and (1-3C)alkyl, includinghydrogen, methyl, ethyl, n-propyl and isopropyl. An example of a valuefor each of Q^(a), Q^(b), Q^(c), Q^(d), Q^(e), Q^(f), Q^(g), Q^(h),Q^(i), Q^(j) and Q^(k) is hydrogen.

In another embodiment, Q^(a), Q^(b), Q^(c), Q^(d), Q^(e), Q^(f), Q^(g),Q^(h), Q^(i), Q^(i) and Q^(k) together with the nitrogen atom and thegroup R^(4b) or R^(4c) to which they are attached, form a 4-6 memberedazacycloalkylene group. For example, Q^(a) and Q^(b) together with thenitrogen atom and the group R^(4b) or R^(4c) to which they are attached,form a piperidin-4-ylene group. By way of illustration, when Qrepresents —N(Q^(a))C(O)— and Q^(a) together with the nitrogen atom andthe group R^(4b) to which it is attached, forms a piperidin-4-ylenegroup, R⁴ is a group of formula:

Similarly, when Q represents —C(O)N(Q^(b))- and Q^(b) together with thenitrogen atom and the group R^(4c) to which it is attached, forms apiperidin-4-ylene group, R⁴ is a group of formula:

In a particular embodiment, R^(4c) is (1-5C)alkylene. Representativeexamples of particular values for R^(4c) are —CH₂—, —(CH₂)₂—, —(CH₂)₃—,—(CH₂)₄—, —(CH₂)₅—; including methylene, ethylene and propylene.

In one embodiment, A² is an optionally substituted (3-7C)cycloalkylenegroup; including a cyclohexylene group, such as cyclohex-1,4-ylene andcyclohex-1,3-ylene; and a cyclopentylene group, such ascyclopent-1,3-ylene.

In another embodiment, A² is an optionally substituted (6-10C)arylenegroup, including a phenylene group, such as phen-1,4-ylene,phen-1,3-ylene and phen-1,2-ylene; and a naphthylene group, such asnaphth-1,4-ylene and napth-1,5-ylene.

In yet another embodiment, A² is an optionally substituted(2-9C)heteroarylene group, including a pyridylene group, such aspyrid-1,4-ylene; a furylene group, such as fur-2,5-ylene andfur-2,4-ylene; a thienylene group, such as thien-2,5-ylene andthien-2,4-ylene; and a pyrrolylene, such as pyrrol-2,5-ylene andpyrrol-2,4-ylene.

In still another embodiment, A² is an optionally substituted(3-6C)heterocyclene group, including a piperidinylene group, such aspiperidin-1,4-ylene; and a pyrrolidinylene group, such aspyrrolidin-2,5-ylene.

In a particular embodiment, A² is optionally substituted phenylene,thienylene, cyclopentylene, cyclohexylene or piperidinylene.

By way of illustration, either A¹ or A² or both can be phenylene, suchas phen-1,4-ylene or phen-1,3-ylene, where the phenylene group isunsubstituted or substituted with from 1 to 4 substituents independentlyselected from halo, (1-4C)alkyl, (1-4C)alkoxy, —S-(1-4C)alkyl,—S(O)-(1-4C)alkyl, —S(O)₂-(1-4C)alkyl, —C(O)O(1-4C)alkyl, carboxy,cyano, hydroxy, nitro, trifluoromethyl and trifluoromethoxy.Representative examples include phen-1,3-ylene, phen-1,4-ylene,4-chlorophen-1,3-ylene, 6-chlorophen-1,3-ylene, 4-methylphen-1,3-ylene,2-fluorophen-1,4-ylene, 2-chlorophen-1,4-ylene, 2-bromophen-1,4-ylene,2-iodophen-1,4-ylene, 2-methylphen-1,4-ylene, 2-methoxyphen-1,4-ylene,2-trifluoromethoxyphen-1,4-ylene, 3-nitrophen-1,4-ylene,3-chlorophen-1,4-ylene, 2,5-difluorophen-1,4-ylene,2,6-dichlorophen-1,4-ylene, 2,6-diiodophen-1,4-ylene,2-chloro-6-methylphen-1,4-ylene, 2-chloro-5-methoxyphen-1,4-ylene,2,3,5,6-tetrafluorophen-1,4-ylene.

Alternatively, A¹ or A² or both can be cyclopentylene or cyclohexylene;wherein the cyclopentylene or cyclohexylene group is unsubstituted orsubstituted with (1-4C)alkyl. Representative examples includecis-cyclopent-1,3-ylene, trans-cyclopent-1,3-ylene,cis-cyclohex-1,4-ylene and trans-cyclohex-1,4-ylene. A¹ or A² or bothcan also be optionally substituted thienylene or piperidinylene, forexample, thien-2,5-ylene or piperidin-1,4-ylene.

In one embodiment, R^(4d) is selected from (1-10C)alkylene,(2-10C)alkenylene and (2-10C)alkynylene wherein the alkylene isunsubstituted or substituted with 1 or 2 substituents independentlyselected from (1-4C)alkyl, hydroxy and phenyl. Representative examplesof particular values for R^(4d) are —(CH₂)—, —(CH₂)₂—, —(CH₂)₃—,—(CH₂)₄—, —(CH₂)₅—, —(CH₂)₆—, —(CH₂)₇—, —(CH₂)₈—, —(CH₂)₉—, —(CH₂)₁₀—and —(CH₂)CH(CH₃)—(CH₂)—C(CH₃)₂—(CH₂)₂—.

In a particular embodiment, the divalent hydrocarbon group is a divalentgroup of the formula: —(R^(4a))_(d)— where R^(4a) is (4-10C)alkylene. Inone aspect of this embodiment, the divalent hydrocarbon group is adivalent group of the formula: —(CH₂)_(j)— where j is 8, 9 or 10.Examples of particular values for the divalent hydrocarbon group in thisembodiment are —(CH₂)₄—, —(CH₂)₅—, —(CH₂)₆—, —(CH₂)₇—, —(CH₂)₈—,—(CH₂)₉, and —(CH₂)₁₀—; including —(CH₂)₈—, —(CH₂)₉, and —(CH₂)₁₀—.

In another particular embodiment, the divalent hydrocarbon group is adivalent group of the formula:—(R^(4a))_(d)-(A²)_(h)-(R^(4d))_(i)—

where R^(4a) is (1-10C)alkylene, such as —(CH₂)—, —(CH₂)₂—, —(CH₂)₃—; A²is (6-10C)arylene, such as phen-1,4-ylene or phen-1,3-ylene, or(2-9C)heteroarylene, such as thien-2,5-ylene or thien-2,4-ylene; andR^(1d) is (1-10C)alkylene, such as —(CH₂)—, —(CH₂)₂—, —(CH₂)₃—. Examplesof particular values for the divalent hydrocarbon group in thisembodiment are —(CH₂)-(phen-1,4-ylene)-(CH₂)—;—(CH₂)-(phen-1,4-ylene)-(CH₂)₂—; —(CH₂)-(phen-1,4-ylene)-(CH₂)₃—;—(CH₂)₂-(phen-1,4-ylene)-(CH₂)—; —(CH₂)₂-(phen-1,4-ylene)-(CH₂)₂—;—(CH₂)₂-(phen-1,4-ylene)-(CH₂)₃—; —(CH₂)₃-(phen-1,4-ylene)-(CH₂)—;—(CH₂)₃-(phen-1,4-ylene)-(CH₂)₂—, —(CH₂)₃-(phen-1,4-ylene)-(CH₂)₃—,—(CH₂)₄-(phen-1,4-ylene)-(CH₂)—; —(CH₂)₄-(phen-1,4-ylene)-(CH₂)₂— and—(CH₂)₄-(phen-1,4-ylene)-(CH₂)₃—.

In yet another particular embodiment, the divalent hydrocarbon group isa divalent group of the formula:—(R^(4a))_(d)-Q-(A²)_(h)-(R^(4d))_(i)—

where Q is —O— or —N(Q^(k))-; Q^(k) is hydrogen or (1-3C)alkyl, such asmethyl or ethyl; R^(4a) is (1-10C)alkylene, such as —(CH₂)—, —(CH₂)₂—,—(CH₂)₃—; A² is (6-10C)arylene, such as phen-1,4-ylene orphen-1,3-ylene, or (2-9C)heteroarylene, such as thien-2,5-ylene orthien-2,4-ylene; and R^(4d) is (1-10C)alkylene, such as —(CH₂)—,—(CH₂)₂—, —(CH₂)₃—. Examples of particular values for the divalenthydrocarbon group in this embodiment are—(CH₂)₂—O-(phen-1,4-ylene)-(CH₂)—; —(CH₂)₂—O-(phen-1,4-ylene)-(CH₂)₂—;—(CH₂)₂—O-(phen-1,4-ylene)-(CH₂)₃—; —(CH₂)₃—O-(phen-1,4-ylene)-(CH₂)—;—(CH₂)₃—O-(phen-1,4-ylene)-(CH₂)₂—; —(CH₂)₃—O-(phen-1,4-ylene)-(CH₂)₃—;—(CH₂)₂—NH-(phen-1,4-ylene)-(CH₂)—; —(CH₂)₂—NH-(phen-1,4-ylene)-(CH₂)₂—;—(CH₂)₂—NH-(phen-1,4-ylene)-(CH₂)₃—; —(CH₂)₃—NH-(phen-1,4-ylene)-(CH₂)—;—(CH₂)₃—NH-(phen-1,4-ylene)-(CH₂)₂— and—(CH₂)₃—NH-(phen-1,4-ylene)-(CH₂)₃—.

In yet another particular embodiment, the divalent hydrocarbon group isa divalent group of the formula:—(R^(4a))_(d)-(A¹)_(e)-(R^(4b))_(f)-Q-(R^(4c))_(g)-(A²)_(h)-(R^(4d))_(i)—

where Q is —N(Q^(a))C(O)— or —C(O)N(Q^(b))-. A particular value for thedivalent hydrocarbon group in this embodiment is the formula:

where m is an integer from 2 to 10; and n is an integer from 2 to 10;provided that m+n is an integer from 4 to 12. In this formula for R⁴, dand g are 1 and e, f, h and i are 0; and R^(4a) is —(CH₂)_(m)—, R^(4c)is —(CH₂)_(n)— and Q is —C(O)NH—. Particular values for m are 2 or 3;and for n, 4, 5 or 6.

Another particular value for the divalent hydrocarbon group is theformula:

where o is an integer from 2 to 7; and p is an integer from 1 to 6;provided that o+p is an integer from 3 to 8. In this formula for R⁴, d,h and i are 1 and e, f and g are 0; and R^(4a) is —(CH₂)_(n)—, A² isphen-1,4-ylene, R^(4d) is —(CH₂)_(p)— and Q is —C(O)NH—. Particularvalues for o are 2 or 3; and for p, 1 or 2. In this embodiment, thephen-1,4-ylene group may be optionally substituted as defined herein forA².

Another particular value for the divalent hydrocarbon group is theformula:

where q is an integer from 2 to 6; r is an integer from 1 to 5; and s isan integer from 1 to 5; provided that q+r+s is an integer from 4 to 8.In this formula for R⁴, d, g, h and i are 1 and e and f are 0; andR^(4a) is —(CH₂)_(q)—, R^(4c) is —(CH₂)_(r)—, A² is 1,4-phenylene,R^(4d) is —(CH₂)_(n)— and Q is —C(O)NH—. Particular values for q are 2or 3; for r, 1 or 2; and for s, 1 or 2. In this embodiment, thephen-1,4-ylene group may be optionally substituted as defined herein forA².

Another particular value for the divalent hydrocarbon group is theformula:

where t is an integer from 2 to 10; and u is an integer from 2 to 10;provided that t+u is an integer from 4 to 12. In this formula for R⁴, dand g are 1 and e, f, h and i are 0; and R^(4a) is —(CH₂)_(t)—, R^(4c)is —(CH₂)_(u)— and Q is —NHC(O)—. Particular values for t are 2 or 3;and for u, 4, 5 or 6.

Another particular value for the divalent hydrocarbon group is theformula:

where v is an integer from 2 to 7; and w is an integer from 1 to 6;provided that v+w is an integer from 3 to 8. In this formula for R⁴, d,h and i are I and e, f and g are 0; and R^(4a) is —(CH₂)_(v)—, A² is1,4-phenylene, R^(4d) is —(CH₂)_(w)— and Q is —NHC(O)—. Particularvalues for v are 2 or 3; and for w, 1 or 2. In this embodiment, thephen-1,4-ylene group may be optionally substituted as defined herein forA².

Another particular value for the divalent hydrocarbon group is theformula:

where x is an integer from 2 to 6; y is an integer from 1 to 5; and z isan integer from 1 to 5; provided that x+y+z is an integer from 4 to 8.In this formula for R⁴, d, g, h and i are 1 and e and f are 0; andR^(4a) is —(CH₂)_(x)—, R^(4c) is —(CH₂)_(y)—, A² is 1,4-phenylene,R^(4d) is —(CH₂)_(z)— and Q is —NHC(O)—. Particular values for x are 2or 3; for y, 1 or 2; and for z, 1 or 2. In this embodiment, thephen-1,4-ylene group may be optionally substituted as defined herein forA².

By way of further illustration, the divalent hydrocarbon group can be agroup selected from:

-   —(CH₂)₇—;-   —(CH₂)₈—;-   —(CH₂)₉—;-   —(CH₂)₁₀—;-   —(CH₂)₁₁—;-   —(CH₂)₂C(O)NH(CH₂)₅—;-   —(CH₂)₂N(CH₃)C(O)(CH₂)₅—;-   —(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂—;-   —(CH₂)₂NHC(O)(phen-1,4-ylene)CH₂—;-   —(CH₂)₂NHC(O)NH(CH₂)₅—;-   —(CH₂)₃NHC(O)NH(CH₂)₅—;-   —(CH₂)₂C(O)NHCH₂(cyclohex-1,3-ylene)CH₂—;-   —(CH₂)₂NHC(O)(cyclopent-1,3-ylene)-;-   —(CH₂)₂NHC(O)NH(phen-1,4-ylene)(CH₂)₂—;-   1-[—(CH₂)₂C(O)](piperidin-4-yl)(CH₂)₂—;-   —(CH₂)₂NHC(O)(trans-cyclohex-1,4-ylene)CH₂—;-   —(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)-;-   —(CH₂)₂NH(phen-1,4-ylene)(CH₂)₂—;-   1-[—(CH₂)₂NHC(O)](piperidin-4-yl)(CH₂)₂—;-   —CH₂(phen-1,4-ylene)NH(phen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)CH₂—;-   —(CH₂)₂C(O)NHCH₂(pyrid-2,6-ylene)CH₂—;-   —(CH₂)₂C(O)NH(cis-cyclohex-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(trans-cyclohex-1,4-ylene)CH₂—;-   —(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)CH₂—;-   —(CH₂)₂N(CH₃)C(O)(phen-1,3-ylene)CH₂—;-   —(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((S)-isomer);-   —(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((R)-isomer);-   2-[(S)-(—CH₂—](pyrrolidin-1-yl)C(O)(CH₂)₄—;-   2-[(S)-(—CH₂—](pyrrolidin-1-yl)C(O)(phen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(4-chlorophen-1,3-ylene)CH₂—;-   —CH₂(2-fluorophen-1,3-ylene)CH₂—;-   —(CH₂)₂C(O)NH(4-methylphen-1,3-ylene)CH₂—;-   —(CH₂)₂C(O)NH(6-chlorophen-1,3-ylene)CH₂—;-   —(CH₂)₂C(O)NH(2-chlorophen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(2,6-dichlorophen-1,4-ylene)CH₂—;-   —(CH₂)₂NHC(O)NHCH₂(phen-1,3-ylene)CH₂—;-   4-[—CH₂—](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)N(CH₂CH₃)(phen-1,4-ylene)CH₂—;-   1-[—(CH₂)₂NHC(O)](piperidin-4-yl)-;-   —(CH₂)₂C(O)NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₂NHC(O)(thien-2,5-ylene)CH₂—;-   —(CH₂)₂N(CH₃)C(O)(3-nitrophen-1,4-ylene)CH₂—;-   —(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)-;-   1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)-;-   5-[—(CH₂)₂NHC(O)](pyrid-2-yl)CH₂—;-   —(CH₂)₂(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₃(thien-2,5-ylene)(CH₂)₃—;-   —(CH₂)₂(phen-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂—;-   —CH₂(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂—;-   1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₂—;-   1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)CH₂—;-   —(CH₂)₂C(O)NH(3-chlorophen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(2-(CF₃O—)phen-1,4-ylene)CH₂—;-   —(CH₂)₃(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₂S(O)₂NH(CH₂)₅—;-   —CH₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₂C(O)NH(2-iodophen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(2-chloro-5-methoxyphen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(2-chloro-6-methylphen-1,4-ylene)CH₂—;-   —(CH₂)₂N(CH₃)S(O)₂(phen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(2-bromophen-1,4-ylene)CH₂—;-   —(CH₂)₃(phen-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₃(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂—;-   1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₃—;-   —(CH₂)₂C(O)NH(2-methoxyphen-1,4-ylene)CH₂—;-   —(CH₂)₅NH(phen-1,4-ylene)(CH₂)₂—;-   4-[—(CH₂)₂—](piperidin-1-yl)(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₂C(O)NH(phen-1,4-ylene)CH(CH₃)CH₂—;-   —(CH₂)₂-(trans-cyclohex-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₂C(O)NH(2-fluorophen-1,4-ylene)CH₂—;-   —(CH₂)₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₂C(O)NH(2,5-difluorophen-1,4-ylene)CH₂—;-   —(CH₂)₂NHC(O)(phen-1,4-ylene)(CH₂)₂—;-   1-[—CH₂(pyrid-2,6-ylene)CH₂](piperidin-4-yl)CH₂—;-   —(CH₂)₃NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₂NH(naphth-1,4-ylene)(CH₂)₂—;-   —(CH₂)₃O(phen-1,4-ylene)CH₂—;-   1-[—(CH₂)₃](piperidin-4-yl)CH₂—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂—;-   —(CH₂)₃(phen-1,4-ylene)NHC(O)(CH₂)₂—;-   —(CH₂)₃O(phen-1,4-ylene)(CH₂)₂—;-   2-[—(CH₂)₂](benzimidazol-5-yl)CH₂—;-   —(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₂—;-   —(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₄—;-   —(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₅—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₂—;-   —(CH₂)₂NHC(O)NH(phen-1,4-ylene)CH₂—;-   —(CH₂)₂N(CH₃)(CH₂)₂(cis-cyclohex-1,4-ylene)-;-   —(CH₂)₂C(O)NH(2,3,5,6-tetrafluorophen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(2,6-diiodophen-1,4-ylene)CH₂—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₃—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₄—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₅—;-   —(CH₂)₂C(O)NHCH₂(phen-1,4-ylene)CH₂—;-   —(CH₂)₂NHC(O)NHCH₂(phen-1,4-ylene)CH₂—;-   —(CH₂)₂C(O)NH(2-methylphen-1,4-ylene)CH₂—;-   1-[—(CH₂)₃O(phen-1,4-ylene)(CH₂)₂](piperidin-4-yl)CH₂—;-   —(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)(CH₂)₂—;-   —(CH₂)₂O(phen-1,3-ylene)CH₂—;-   —(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,4-ylene)CH₂—;-   —(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,3-ylene)CH₂—;-   —(CH₂)₂N(CH₃)C(O)(fur-2,5-ylene)CH₂—;-   —(CH₂)₂N(CH₃)C(O)(thien-2,5-ylene)CH₂—;-   —(CH₂)₂O(phen-1,4-ylene)O(CH₂)₂—;-   —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂—;-   —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,2-ylene)CH₂—;-   —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,3-ylene)CH₂—;-   —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,4-ylene)CH₂—;-   —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂—;-   —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,2-ylene)CH₂—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,3-ylene)CH₂—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,4-ylene)CH₂—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)(fur-2,5-ylene)CH₂—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)(thien-2,5-ylene)CH₂—;-   —(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂—;-   —(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂—;-   —(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,2-ylene)CH₂—;-   —(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,3-ylene)CH₂—;-   —(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,4-ylene)CH₂—;-   —(CH₂)₂(phen-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂—;-   —(CH₂)₂(phen-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂—;-   —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂—;-   —(CH₂)₃O(phen-1,3-ylene)CH₂—;-   —CH₂CH(OH)CH₂NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₄NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂NHC(O)CH₂—;-   —(CH₂)₂C(O)NH(phen-1,4-ylene)(CH₂)₂NHC(O)CH₂—;-   —(CH₂)₂C(O)NHCH₂(trans-cyclohex-1,4-ylene)CH₂—;-   —(CH₂)₂NHC(O)(CH₂)₅—;-   —(CH₂)₂O(phen-1,3-ylene)O(CH₂)₂—;-   —(CH₂)₂O(phen-1,2-ylene)O(CH₂)₂—;-   —CH₂(phen-1,2-ylene)O(phen-1,2-ylene)CH₂—;-   —(CH₂)₂C(O)NH(CH₂)₆—;-   —(CH₂)₃(phen-1,4-ylene)(CH₂)₃—;-   —(CH₂)₃(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₄(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₃(furan-2,5-ylene)(CH₂)₃—;-   —(CH₂)₂N(CH₃)C(O)NH(phen-1,4-ylene)(CH₂)₂—;-   4-[—(CH₂)₂](piperidin-1-yl)C(O)NH(phen-1,4-ylene)(CH₂)₂—;-   —(CH₂)₃(phen-1,3-ylene)(CH₂)₃—;-   —(CH₂)₃(tetrahydrofuran-2,5-ylene)(CH₂)₃—; and-   —(CH₂)₂O(phen-1,4-ylene)C(O)(CH₂)₂—.    Representative Subgeneric Groupings

The following subgeneric formulae and groupings are intended to providerepresentative examples of various aspects and embodiments of thisinvention and as such, they are not intended to exclude otherembodiments or to limit the scope of this invention unless otherwiseindicated.

A particular group of compounds of formula I are those disclosed in U.S.Provisional Application No. 60/524,234, filed on Nov. 21, 2003. Thisgroup includes compounds of formula Ia; wherein:

a is 0 or an integer of from 1 to 3;

each R¹ is independently selected from the group consisting of(1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano,halo, —OR^(1a), —C(O)OR^(1b), SR^(1c), —S(O)R^(1d), —S(O)₂R^(1e), and—NR^(1f)R^(1g);

each of R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f) and R^(1g) isindependently hydrogen or (1-4C)alkyl;

b is 0 or an integer of from 1 to 3;

each R² is independently selected from the group consisting of(1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano,halo, —OR^(2a), —C(O)OR^(2b), SR^(2c), —S(O)R^(2d), —S(O)₂R^(2e), and—NR^(2f)R^(2g);

each of R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f) and R^(2g) isindependently hydrogen or (1-4C)alkyl;

W is attached to the 3- or 4-position with respect to the nitrogen atomin the piperidine ring, and represents O or NW^(a);

W^(a) is hydrogen or (1-4C)alkyl;

c is 0 or an integer of from 1 to 4;

each R³ is a substituent on carbon independently selected from the groupconsisting of (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl,(3-6C)cycloalkyl, cyano, halo, —OR^(3a), —C(O)OR^(3b), SR^(3c),—S(O)R^(3d), —S(O)₂R^(3e), and —NR^(3f)R^(3g);

each of R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f) and R^(3g) isindependently hydrogen or (1-4C)alkyl;

R⁴ is a divalent group of the formula:—(R^(4a))_(d)-(A¹)_(e)-(R^(4b))_(f)-Q-(R^(4c))_(g)-(A²)_(h)-(R^(4d))_(i)—wherein

d, e, f, g, h and i are each independently selected from 0 and 1;

R^(4a), R^(4b), R^(4c) and R^(4d) are each independently selected fromthe group consisting of (1-10C)alkylene, (2-10C)alkenylene and(2-10C)alkynylene wherein each alkylene, alkenylene or alkynylene groupis unsubstituted or substituted with from 1 to 5 substituentsindependently selected from the group consisting of (1-4C)alkyl, fluoro,hydroxy, phenyl and phenyl(1-4C)-alkyl;

A¹ and A² are each independently selected from (3-7C)cycloalkylene,(6-10C)arylene, (2-9C)heteroarylene and (3-6C)heterocyclene; whereineach cycloalkylene is unsubstituted or substituted with from 1 to 4substitutents selected independently from (1-4C)alkyl and each arylene,heteroarylene or heterocyclene group is unsubstituted or substitutedwith from 1 to 4 substituents independently selected from the groupconsisting of halogen, (1-4C)alkyl and (1-4C)alkoxy;

Q is selected from the group consisting of a bond, —O—, —C(O)O—,—OC(O)—, —S—, —S(O)—, —S(O)₂—, —N(Q^(a))C(O)—, —C(O)N(Q^(b))-,—N(Q^(c))S(O)₂—, —S(O)₂N(Q^(d))-, —N(Q^(e))C(O)N(Q^(f))-,—N(Q^(g))S(O)₂N(Q^(h))-, —OC(O)N(Q^(i))-, —N(Q^(j))C(O)O— and N(Q^(k))-;

Q^(a), Q^(b), Q^(c), Q^(d), Q^(e), Q^(f), Q^(g), Q^(h), Q^(i), Q^(j) andQ^(k) are each independently selected from the group consisting ofhydrogen, (1-6C)alkyl, A³ and (1-4C)alkylene-A⁴; wherein the alkyl groupis unsubstituted or substituted with from 1 to 3 substituentsindependently selected from fluoro, hydroxy and (1-4C)alkoxy; ortogether with the nitrogen atom and the group R^(4b) or R^(4c) to whichthey are attached, form a 4-6 membered azacycloalkylene group;

A³ and A⁴ are each independently selected from (3-6C)cycloalkyl,(6-10C)aryl, (2-9C)heteroaryl and (3-6C)heterocyclyl; wherein eachcycloalkyl is unsubstituted or substituted with from 1 to 4substitutents selected independently from (1-4C)alkyl and each aryl,heteroaryl or heterocyclyl group is unsubstituted or substituted withfrom 1 to 4 substituents independently selected from the groupconsisting of halogen, (1-4C)alkyl and (1-4C)alkoxy;

provided that the number of contiguous atoms in the shortest chainbetween the two nitrogen atoms to which R⁴ is attached is in the rangeof from 4 to 14;

R⁵ represents hydrogen or (1-4C)alkyl;

R⁶ represents hydrogen or hydroxyl; and

one of G¹ and G² represents NH and the other represents S, NH, O or CH₂;

or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof.

Another particular group of compounds of formula I are those where: a is0; b is 0; c is 0; d is 0; m is 2; W is O; W is attached at the4-position of the piperidinyl ring; R⁵ is hydrogen; and R⁴, R⁶, G¹ andG² are as defined herein; or a pharmaceutically acceptable salt orsolvate or stereoisomer thereof.

Another particular group of compounds of formula I are those where: a is0; b is 0; c is 0; d is 1; m is 2; W is O; W is attached at the4-position of the piperidinyl ring; R⁵ is hydrogen; R^(7a) and R^(7b)are hydrogen; and R⁴, R⁶, G¹ and G² are as defined herein; or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

Still another particular group of compounds of formula I are thosewherein: a is 0; b is 0; c is 0; d is 0; m is 2; W is NH; W is attachedat the 4-position of the piperidinyl ring; R⁵ is hydrogen; and R⁴, R⁶,G¹ and G² are as defined herein; or a pharmaceutically acceptable saltor solvate or stereoisomer thereof.

Another particular group of compounds of formula I are those of formulaIa as defined herein; or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

Another particular group of compounds of formula I are those of formulaIb as defined herein; or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

Another particular group of compounds of formula I are those of formulaIc as defined herein; or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

Another particular group of compounds of formula I are those of formulaId as defined herein; or a pharmaceutically acceptable salt or solvateor stereoisomer thereof.

Another particular group of compounds of formula I are those of formulaIa, Ib, Ic or Id as defined herein, wherein the piperidinyl ring issubstituted at the 4-position with a methyl group; or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof.

Further examples of representative compounds of this invention arecompounds of formula Ie:

wherein W, R⁴ and R⁶ are as defined in Table I; or a pharmaceuticallyacceptable salt or solvate thereof.

TABLE I Ex. W R⁴ R⁶ 1 O —(CH₂)₉— H 2 O —(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂—H 3 NH —(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂— H 4 O—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂CH₂— H 5 O—(CH₂)₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 6 O—(CH₂)₂(phen-1,4-ylene)(CH₂)₂— H 7 O—(CH₂)₂(phen-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂— H 8 O—(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂— H 9 O—(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂— H 10 O—(CH₂)₂(phen-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂— H 11 O—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,2-ylene)CH₂— H 12 O—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,3-ylene)CH₂— H 13 O—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,4-ylene)CH₂— H 14 O—(CH₂)₂-(trans-cyclohex-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 15 O—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₂— H 16 O—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₄— H 17 O—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₅— H 18 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂— H 19 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂— H 20 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂— H 21 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂— H 22 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,2- H ylene)CH₂— 23 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,3- H ylene)CH₂— 24 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,4- H ylene)CH₂— 25 O—(CH₂)₂C(O)N(CH₂CH₃)(phen-1,4-ylene)CH₂— H 26 O—(CH₂)₂C(O)NH(2-(CF₃O—)phen-1,4-ylene)CH₂— H 27 O—(CH₂)₂C(O)NH(2,3,5,6-tetrafluorophen-1,4-ylene)CH₂— H 28 O—(CH₂)₂C(O)NH(2,5-difluorophen-1,4-ylene)CH₂— H 29 O—(CH₂)₂C(O)NH(2,6-dichlorophen-1,4-ylene)CH₂— H 30 O—(CH₂)₂C(O)NH(2,6-diiodophen-1,4-ylene)CH₂— H 31 O—(CH₂)₂C(O)NH(2-bromophen-1,4-ylene)CH₂— H 32 O—(CH₂)₂C(O)NH(2-chloro-5-methoxyphen-1,4-ylene)CH₂— H 33 O—(CH₂)₂C(O)NH(2-chloro-6-methylphen-1,4-ylene)CH₂— H 34 O—(CH₂)₂C(O)NH(2-chlorophen-1,4-ylene)CH₂— H 35 O—(CH₂)₂C(O)NH(2-fluorophen-1,4-ylene)CH₂— H 36 O—(CH₂)₂C(O)NH(2-iodophen-1,4-ylene)CH₂— H 37 O—(CH₂)₂C(O)NH(2-methoxyphen-1,4-ylene)CH₂— H 38 O—(CH₂)₂C(O)NH(2-methylphen-1,4-ylene)CH₂— H 39 O—(CH₂)₂C(O)NH(3-chlorophen-1,4-ylene)CH₂— H 40 O—(CH₂)₂C(O)NH(4-chlorophen-1,3-ylene)CH₂— H 41 O—(CH₂)₂C(O)NH(4-methylphen-1,3-ylene)CH₂— H 42 O—(CH₂)₂C(O)NH(6-chlorophen-1,3-ylene)CH₂— H 43 O —(CH₂)₂C(O)NH(CH₂)₅— H44 O —(CH₂)₂C(O)NH(CH₂)₆— H 45 O—(CH₂)₂C(O)NH(cis-cyclohex-1,4-ylene)CH₂— H 46 O—(CH₂)₂C(O)NH(phen-1,4-ylene)(CH₂)₂NHC(O)CH₂— H 47 O—(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((S)-isomer) H 48 O—(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((R)-isomer) H 49 O—(CH₂)₂C(O)NH(phen-1,4-ylene)CH(CH₃)CH₂— H 50 O—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂NHC(O)CH₂— H 51 O—(CH₂)₂C(O)NH(trans-cyclohex-1,4-ylene)CH₂— H 52 O—(CH₂)₂C(O)NHCH₂(cyclohex-1,3-ylene)CH₂— H 53 O—(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)(CH₂)₂— H 54 O—(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)CH₂— H 55 O—(CH₂)₂C(O)NHCH₂(phen-1,4-ylene)CH₂— H 56 O—(CH₂)₂C(O)NHCH₂(pyrid-2,6-ylene)CH₂— H 57 O—(CH₂)₂C(O)NHCH₂(trans-cyclohex-1,4-ylene)CH₂— H 58 O—(CH₂)₂N(CH₃)(CH₂)₂(cis-cyclohex-1,4-ylene)- H 59 O—(CH₂)₂N(CH₃)C(O)(3-nitrophen-1,4-ylene)CH₂— H 60 O—(CH₂)₂N(CH₃)C(O)(CH₂)₅— H 61 O —(CH₂)₂N(CH₃)C(O)(fur-2,5-ylene)CH₂— H62 O —(CH₂)₂N(CH₃)C(O)(phen-1,3-ylene)CH₂— H 63 O—(CH₂)₂N(CH₃)C(O)(thien-2,5-ylene)CH₂— H 64 O—(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)- H 65 O—(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)CH₂— H 66 O—(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,3-ylene)CH₂— H 67 O—(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,4-ylene)CH₂— H 68 O—(CH₂)₂N(CH₃)C(O)NH(phen-1,4-ylene)(CH₂)₂— H 69 O—(CH₂)₂N(CH₃)S(O)₂(phen-1,4-ylene)CH₂— H 70 O—(CH₂)₂NH(naphth-1,4-ylene)(CH₂)₂— H 71 O—(CH₂)₂NH(phen-1,4-ylene)(CH₂)₂— H 72 O —(CH₂)₂NHC(O)(CH₂)₅— H 73 O—(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)- H 74 O—(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)CH₂— H 75 O—(CH₂)₂NHC(O)(phen-1,4-ylene)(CH₂)₂— H 76 O—(CH₂)₂NHC(O)(phen-1,4-ylene)CH₂— H 77 O—(CH₂)₂NHC(O)(thien-2,5-ylene)CH₂— H 78 O—(CH₂)₂NHC(O)(trans-cyclohex-1,4-ylene)CH₂— H 79 O—(CH₂)₂NHC(O)NH(CH₂)₅— H 80 O —(CH₂)₂NHC(O)NH(phen-1,4-ylene)(CH₂)₂— H81 O —(CH₂)₂NHC(O)NH(phen-1,4-ylene)CH₂— H 82 O—(CH₂)₂NHC(O)NHCH₂(phen-1,3-ylene)CH₂— H 83 O—(CH₂)₂NHC(O)NHCH₂(phen-1,4-ylene)CH₂— H 84 O—(CH₂)₂O(phen-1,2-ylene)O(CH₂)₂— H 85 O —(CH₂)₂O(phen-1,3-ylene)CH₂— H86 O —(CH₂)₂O(phen-1,3-ylene)O(CH₂)₂— H 87 O—(CH₂)₂O(phen-1,4-ylene)C(O)(CH₂)₂— H 88 O—(CH₂)₂O(phen-1,4-ylene)O(CH₂)₂— H 89 O —(CH₂)₂S(O)₂NH(CH₂)₅— H 90 O—(CH₂)₃(furan-2,5-ylene)(CH₂)₃— H 91 O—(CH₂)₃(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 92 O—(CH₂)₃(phen-1,3-ylene)(CH₂)₃— H 93 O—(CH₂)₃(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 94 O—(CH₂)₃(phen-1,4-ylene)(CH₂)₂— H 95 O —(CH₂)₃(phen-1,4-ylene)(CH₂)₃— H96 O —(CH₂)₃(phen-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 97 O—(CH₂)₃(phen-1,4-ylene)NHC(O)(CH₂)₂— H 98 O—(CH₂)₃(tetrahydrofuran-2,5-ylene)(CH₂)₃— H 99 O—(CH₂)₃(thien-2,5-ylene)(CH₂)₃— H 100 O —(CH₂)₃NH(phen-1,4-ylene)(CH₂)₂—H 101 O —(CH₂)₃NHC(O)NH(CH₂)₅— H 102 O —(CH₂)₃O(phen-1,3-ylene)CH₂— H103 O —(CH₂)₃O(phen-1,4-ylene)(CH₂)₂— H 104 O—(CH₂)₃O(phen-1,4-ylene)CH₂— H 105 O —(CH₂)₄(phen-1,4-ylene)(CH₂)₂— H106 O —(CH₂)₄NH(phen-1,4-ylene)(CH₂)₂— H 107 O—(CH₂)₅NH(phen-1,4-ylene)(CH₂)₂— H 108 O —(CH₂)₇— H 109 O —(CH₂)₈— H 110O 1-[—(CH₂)₂C(O)](piperidin-4-yl)(CH₂)₂— H 111 O1-[—(CH₂)₂NHC(O)](piperidin-4-yl)- H 112 O1-[—(CH₂)₂NHC(O)](piperidin-4-yl)(CH₂)₂— H 113 O1-[—(CH₂)₃](piperidin-4-yl)CH₂— H 114 O1-[—(CH₂)₃O(phen-1,4-ylene)(CH₂)₂](piperidin-4-yl)CH₂— H 115 O1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)- H 116 O1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₂— H 117 O1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₃— H 118 O1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)CH₂— H 119 O1-[—CH₂(pyrid-2,6-ylene)CH₂](piperidin-4-yl)CH₂— H 120 O2-[—(CH₂)₂](benzimidazol-5-yl)CH₂— H 121 O2-[(S)—(—CH₂—](pyrrolidin-1-yl)C(O)(CH₂)₄— H 122 O2-[(S)—(—CH₂—](pyrrolidin-1-yl)C(O)(phen-1,4-ylene)CH₂— H 123 O4-[—(CH₂)₂—](piperidin-1-yl)(phen-1,4-ylene)(CH₂)₂— H 124 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₂— H 125 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₃— H 126 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₄— H 127 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₅— H 128 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(fur-2,5-ylene)CH₂— H 129 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂— H 130 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(thien-2,5-ylene)CH₂— H 131 O4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,2-ylene)CH₂— H 132 O4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,3-ylene)CH₂— H 133 O4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,4-ylene)CH₂— H 134 O4-[—(CH₂)₂](piperidin-1-yl)C(O)NH(phen-1,4-ylene)(CH₂)₂— H 135 O4-[—CH₂—](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂— H 136 O5-[—(CH₂)₂NHC(O)](pyrid-2-yl)CH₂— H 137 O—CH₂(2-fluorophen-1,3-ylene)CH₂— H 138 O—CH₂(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 139 O—CH₂(phen-1,2-ylene)O(phen-1,2-ylene)CH₂— H 140 O—CH₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 141 O—CH₂(phen-1,4-ylene)NH(phen-1,4-ylene)CH₂— H 142 O—CH₂CH(OH)CH₂NH(phen-1,4-ylene)(CH₂)₂— H 143 O —(CH₂)₉— OH 144 O—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂— OH 145 O—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂CH₂— OH 146 O—(CH₂)₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 147 O—(CH₂)₂(phen-1,4-ylene)(CH₂)₂— OH 148 O—(CH₂)₂(phen-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂— OH 149 O—(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂— OH 150 O—(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂— OH 151 O—(CH₂)₂(phen-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂— OH 152 O—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,2-ylene)CH₂— OH 153 O—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,3-ylene)CH₂— OH 154 O—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,4-ylene)CH₂— OH 155 O—(CH₂)₂-(trans-cyclohex-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 156 O—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₂— OH 157 O—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₄— OH 158 O—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₅— OH 159 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂— OH 160 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂— OH 161 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂— OH 162 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂— OH 163 O—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,2- OH ylene)CH₂— 164O —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,3- OH ylene)CH₂—165 O —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,4- OHylene)CH₂— 166 O —(CH₂)₂C(O)N(CH₂CH₃)(phen-1,4-ylene)CH₂— OH 167 O—(CH₂)₂C(O)NH(2-(CF₃O—)phen-1,4-ylene)CH₂— OH 168 O—(CH₂)₂C(O)NH(2,3,5,6-tetrafluorophen-1,4-ylene)CH₂— OH 169 O—(CH₂)₂C(O)NH(2,5-difluorophen-1,4-ylene)CH₂— OH 170 O—(CH₂)₂C(O)NH(2,6-dichlorophen-1,4-ylene)CH₂— OH 171 O—(CH₂)₂C(O)NH(2,6-diiodophen-1,4-ylene)CH₂— OH 172 O—(CH₂)₂C(O)NH(2-bromophen-1,4-ylene)CH₂— OH 173 O—(CH₂)₂C(O)NH(2-chloro-5-methoxyphen-1,4-ylene)CH₂— OH 174 O—(CH₂)₂C(O)NH(2-chloro-6-methylphen-1,4-ylene)CH₂— OH 175 O—(CH₂)₂C(O)NH(2-chlorophen-1,4-ylene)CH₂— OH 176 O—(CH₂)₂C(O)NH(2-fluorophen-1,4-ylene)CH₂— OH 177 O—(CH₂)₂C(O)NH(2-iodophen-1,4-ylene)CH₂— OH 178 O—(CH₂)₂C(O)NH(2-methoxyphen-1,4-ylene)CH₂— OH 179 O—(CH₂)₂C(O)NH(2-methylphen-1,4-ylene)CH₂— OH 180 O—(CH₂)₂C(O)NH(3-chlorophen-1,4-ylene)CH₂— OH 181 O—(CH₂)₂C(O)NH(4-chlorophen-1,3-ylene)CH₂— OH 182 O—(CH₂)₂C(O)NH(4-methylphen-1,3-ylene)CH₂— OH 183 O—(CH₂)₂C(O)NH(6-chlorophen-1,3-ylene)CH₂— OH 184 O —(CH₂)₂C(O)NH(CH₂)₅—OH 185 O —(CH₂)₂C(O)NH(CH₂)₆— OH 186 O—(CH₂)₂C(O)NH(cis-cyclohex-1,4-ylene)CH₂— OH 187 O—(CH₂)₂C(O)NH(phen-1,4-ylene)(CH₂)₂NHC(O)CH₂— OH 188 O—(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((S)-isomer) OH 189 O—(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((R)-isomer) OH 190 O—(CH₂)₂C(O)NH(phen-1,4-ylene)CH(CH₃)CH₂— OH 191 O—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂NHC(O)CH₂— OH 192 O—(CH₂)₂C(O)NH(trans-cyclohex-1,4-ylene)CH₂— OH 193 O—(CH₂)₂C(O)NHCH₂(cyclohex-1,3-ylene)CH₂— OH 194 O—(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)(CH₂)₂— OH 195 O—(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)CH₂— OH 196 O—(CH₂)₂C(O)NHCH₂(phen-1,4-ylene)CH₂— OH 197 O—(CH₂)₂C(O)NHCH₂(pyrid-2,6-ylene)CH₂— OH 198 O—(CH₂)₂C(O)NHCH₂(trans-cyclohex-1,4-ylene)CH₂— OH 199 O—(CH₂)₂N(CH₃)(CH₂)₂(cis-cyclohex-1,4-ylene)- OH 200 O—(CH₂)₂N(CH₃)C(O)(3-nitrophen-1,4-ylene)CH₂— OH 201 O—(CH₂)₂N(CH₃)C(O)(CH₂)₅— OH 202 O —(CH₂)₂N(CH₃)C(O)(fur-2,5-ylene)CH₂—OH 203 O —(CH₂)₂N(CH₃)C(O)(phen-1,3-ylene)CH₂— OH 204 O—(CH₂)₂N(CH₃)C(O)(thien-2,5-ylene)CH₂— OH 205 O—(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)- OH 206 O—(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)CH₂— OH 207 O—(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,3-ylene)CH₂— OH 208 O—(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,4-ylene)CH₂— OH 209 O—(CH₂)₂N(CH₃)C(O)NH(phen-1,4-ylene)(CH₂)₂— OH 210 O—(CH₂)₂N(CH₃)S(O)₂(phen-1,4-ylene)CH₂— OH 211 O—(CH₂)₂NH(naphth-1,4-ylene)(CH₂)₂— OH 212 O—(CH₂)₂NH(phen-1,4-ylene)(CH₂)₂— OH 213 O —(CH₂)₂NHC(O)(CH₂)₅— OH 214 O—(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)- OH 215 O—(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)CH₂— OH 216 O—(CH₂)₂NHC(O)(phen-1,4-ylene)(CH₂)₂— OH 217 O—(CH₂)₂NHC(O)(phen-1,4-ylene)CH₂— OH 218 O—(CH₂)₂NHC(O)(thien-2,5-ylene)CH₂— OH 219 O—(CH₂)₂NHC(O)(trans-cyclohex-1,4-ylene)CH₂— OH 220 O—(CH₂)₂NHC(O)NH(CH₂)₅— OH 221 O —(CH₂)₂NHC(O)NH(phen-1,4-ylene)(CH₂)₂—OH 222 O —(CH₂)₂NHC(O)NH(phen-1,4-ylene)CH₂— OH 223 O—(CH₂)₂NHC(O)NHCH₂(phen-1,3-ylene)CH₂— OH 224 O—(CH₂)₂NHC(O)NHCH₂(phen-1,4-ylene)CH₂— OH 225 O—(CH₂)₂O(phen-1,2-ylene)O(CH₂)₂— OH 226 O —(CH₂)₂O(phen-1,3-ylene)CH₂—OH 227 O —(CH₂)₂O(phen-1,3-ylene)O(CH₂)₂— OH 228 O—(CH₂)₂O(phen-1,4-ylene)C(O)(CH₂)₂— OH 229 O—(CH₂)₂O(phen-1,4-ylene)O(CH₂)₂— OH 230 O —(CH₂)₂S(O)₂NH(CH₂)₅— OH 231 O—(CH₂)₃(furan-2,5-ylene)(CH₂)₃— OH 232 O—(CH₂)₃(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 233 O—(CH₂)₃(phen-1,3-ylene)(CH₂)₃— OH 234 O—(CH₂)₃(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 235 O—(CH₂)₃(phen-1,4-ylene)(CH₂)₂— OH 236 O —(CH₂)₃(phen-1,4-ylene)(CH₂)₃—OH 237 O —(CH₂)₃(phen-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 238 O—(CH₂)₃(phen-1,4-ylene)NHC(O)(CH₂)₂— OH 239 O—(CH₂)₃(tetrahydrofuran-2,5-ylene)(CH₂)₃— OH 240 O—(CH₂)₃(thien-2,5-ylene)(CH₂)₃— OH 241 O—(CH₂)₃NH(phen-1,4-ylene)(CH₂)₂— OH 242 O —(CH₂)₃NHC(O)NH(CH₂)₅— OH 243O —(CH₂)₃O(phen-1,3-ylene)CH₂— OH 244 O —(CH₂)₃O(phen-1,4-ylene)(CH₂)₂—OH 245 O —(CH₂)₃O(phen-1,4-ylene)CH₂— OH 246 O—(CH₂)₄(phen-1,4-ylene)(CH₂)₂— OH 247 O —(CH₂)₄NH(phen-1,4-ylene)(CH₂)₂—OH 248 O —(CH₂)₅NH(phen-1,4-ylene)(CH₂)₂— OH 249 O —(CH₂)₇— OH 250 O—(CH₂)₈— OH 251 O 1-[—(CH₂)₂C(O)](piperidin-4-yl)(CH₂)₂— OH 252 O1-[—(CH₂)₂NHC(O)](piperidin-4-yl)- OH 253 O1-[—(CH₂)₂NHC(O)](piperidin-4-yl)(CH₂)₂— OH 254 O1-[—(CH₂)₃](piperidin-4-yl)CH₂— OH 255 O1-[—(CH₂)₃O(phen-1,4-ylene)(CH₂)₂](piperidin-4-yl)CH₂— OH 256 O1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)- OH 257 O1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₂— OH 258 O1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₃— OH 259 O1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)CH₂— OH 260 O1-[—CH₂(pyrid-2,6-ylene)CH₂](piperidin-4-yl)CH₂— OH 261 O2-[—(CH₂)₂](benzimidazol-5-yl)CH₂— OH 262 O2-[(S)—(—CH₂—](pyrrolidin-1-yl)C(O)(CH₂)₄— OH 263 O2-[(S)—(—CH₂—](pyrrolidin-1-yl)C(O)(phen-1,4-ylene)CH₂— OH 264 O4-[—(CH₂)₂—](piperidin-1-yl)(phen-1,4-ylene)(CH₂)₂— OH 265 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₂— OH 266 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₃— OH 267 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₄— OH 268 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₅— OH 269 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(fur-2,5-ylene)CH₂— OH 270 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂— OH 271 O4-[—(CH₂)₂](piperidin-1-yl)C(O)(thien-2,5-ylene)CH₂— OH 272 O4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,2-ylene)CH₂— OH 273 O4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,3-ylene)CH₂— OH 274 O4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,4-ylene)CH₂— OH 275 O4-[—(CH₂)₂](piperidin-1-yl)C(O)NH(phen-1,4-ylene)(CH₂)₂— OH 276 O4-[—CH₂—](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂— OH 277 O5-[—(CH₂)₂NHC(O)](pyrid-2-yl)CH₂— OH 278 O—CH₂(2-fluorophen-1,3-ylene)CH₂— OH 279 O—CH₂(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 280 O—CH₂(phen-1,2-ylene)O(phen-1,2-ylene)CH₂— OH 281 O—CH₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 282 O—CH₂(phen-1,4-ylene)NH(phen-1,4-ylene)CH₂— OH 283 O—CH₂CH(OH)CH₂NH(phen-1,4-ylene)(CH₂)₂— OH 284 NH —(CH₂)₉— H 285 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂CH₂— H 286 NH—(CH₂)₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 287 NH—(CH₂)₂(phen-1,4-ylene)(CH₂)₂— H 288 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂— H 289 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂— H 290 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂— H 291 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂— H 292 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,2-ylene)CH₂— H 293 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,3-ylene)CH₂— H 294 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,4-ylene)CH₂— H 295 NH—(CH₂)₂-(trans-cyclohex-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 296 NH—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₂— H 297 NH—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₄— H 298 NH—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₅— H 299 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂— H 300 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂— H 301 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂— H 302 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂— H 303 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,2- H ylene)CH₂— 304NH —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,3- H ylene)CH₂—305 NH —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,4- Hylene)CH₂— 306 NH —(CH₂)₂C(O)N(CH₂CH₃)(phen-1,4-ylene)CH₂— H 307 NH—(CH₂)₂C(O)NH(2-(CF₃O—)phen-1,4-ylene)CH₂— H 308 NH—(CH₂)₂C(O)NH(2,3,5,6-tetrafluorophen-1,4-ylene)CH₂— H 309 NH—(CH₂)₂C(O)NH(2,5-difluorophen-1,4-ylene)CH₂— H 310 NH—(CH₂)₂C(O)NH(2,6-dichlorophen-1,4-ylene)CH₂— H 311 NH—(CH₂)₂C(O)NH(2,6-diiodophen-1,4-ylene)CH₂— H 312 NH—(CH₂)₂C(O)NH(2-bromophen-1,4-ylene)CH₂— H 313 NH—(CH₂)₂C(O)NH(2-chloro-5-methoxyphen-1,4-ylene)CH₂— H 314 NH—(CH₂)₂C(O)NH(2-chloro-6-methylphen-1,4-ylene)CH₂— H 315 NH—(CH₂)₂C(O)NH(2-chlorophen-1,4-ylene)CH₂— H 316 NH—(CH₂)₂C(O)NH(2-fluorophen-1,4-ylene)CH₂— H 317 NH—(CH₂)₂C(O)NH(2-iodophen-1,4-ylene)CH₂— H 318 NH—(CH₂)₂C(O)NH(2-methoxyphen-1,4-ylene)CH₂— H 319 NH—(CH₂)₂C(O)NH(2-methylphen-1,4-ylene)CH₂— H 320 NH—(CH₂)₂C(O)NH(3-chlorophen-1,4-ylene)CH₂— H 321 NH—(CH₂)₂C(O)NH(4-chlorophen-1,3-ylene)CH₂— H 322 NH—(CH₂)₂C(O)NH(4-methylphen-1,3-ylene)CH₂— H 323 NH—(CH₂)₂C(O)NH(6-chlorophen-1,3-ylene)CH₂— H 324 NH —(CH₂)₂C(O)NH(CH₂)₅—H 325 NH —(CH₂)₂C(O)NH(CH₂)₆— H 326 NH—(CH₂)₂C(O)NH(cis-cyclohex-1,4-ylene)CH₂— H 327 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)(CH₂)₂NHC(O)CH₂— H 328 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((S)-isomer) H 329 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((R)-isomer) H 330 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)CH(CH₃)CH₂— H 331 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂NHC(O)CH₂— H 332 NH—(CH₂)₂C(O)NH(trans-cyclohex-1,4-ylene)CH₂— H 333 NH—(CH₂)₂C(O)NHCH₂(cyclohex-1,3-ylene)CH₂— H 334 NH—(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)(CH₂)₂— H 335 NH—(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)CH₂— H 336 NH—(CH₂)₂C(O)NHCH₂(phen-1,4-ylene)CH₂— H 337 NH—(CH₂)₂C(O)NHCH₂(pyrid-2,6-ylene)CH₂— H 338 NH—(CH₂)₂C(O)NHCH₂(trans-cyclohex-1,4-ylene)CH₂— H 339 NH—(CH₂)₂N(CH₃)(CH₂)₂(cis-cyclohex-1,4-ylene)- H 340 NH—(CH₂)₂N(CH₃)C(O)(3-nitrophen-1,4-ylene)CH₂— H 341 NH—(CH₂)₂N(CH₃)C(O)(CH₂)₅— H 342 NH —(CH₂)₂N(CH₃)C(O)(fur-2,5-ylene)CH₂— H343 NH —(CH₂)₂N(CH₃)C(O)(phen-1,3-ylene)CH₂— H 344 NH—(CH₂)₂N(CH₃)C(O)(thien-2,5-ylene)CH₂— H 345 NH—(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)- H 346 NH—(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)CH₂— H 347 NH—(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,3-ylene)CH₂— H 348 NH—(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,4-ylene)CH₂— H 349 NH—(CH₂)₂N(CH₃)C(O)NH(phen-1,4-ylene)(CH₂)₂— H 350 NH—(CH₂)₂N(CH₃)S(O)₂(phen-1,4-ylene)CH₂— H 351 NH—(CH₂)₂NH(naphth-1,4-ylene)(CH₂)₂— H 352 NH—(CH₂)₂NH(phen-1,4-ylene)(CH₂)₂— H 353 NH —(CH₂)₂NHC(O)(CH₂)₅— H 354 NH—(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)- H 355 NH—(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)CH₂— H 356 NH—(CH₂)₂NHC(O)(phen-1,4-ylene)(CH₂)₂— H 357 NH—(CH₂)₂NHC(O)(phen-1,4-ylene)CH₂— H 358 NH—(CH₂)₂NHC(O)(thien-2,5-ylene)CH₂— H 359 NH—(CH₂)₂NHC(O)(trans-cyclohex-1,4-ylene)CH₂— H 360 NH—(CH₂)₂NHC(O)NH(CH₂)₅— H 361 NH —(CH₂)₂NHC(O)NH(phen-1,4-ylene)(CH₂)₂— H362 NH —(CH₂)₂NHC(O)NH(phen-1,4-ylene)CH₂— H 363 NH—(CH₂)₂NHC(O)NHCH₂(phen-1,3-ylene)CH₂— H 364 NH—(CH₂)₂NHC(O)NHCH₂(phen-1,4-ylene)CH₂— H 365 NH—(CH₂)₂O(phen-1,2-ylene)O(CH₂)₂— H 366 NH —(CH₂)₂O(phen-1,3-ylene)CH₂— H367 NH —(CH₂)₂O(phen-1,3-ylene)O(CH₂)₂— H 368 NH—(CH₂)₂O(phen-1,4-ylene)C(O)(CH₂)₂— H 369 NH—(CH₂)₂O(phen-1,4-ylene)O(CH₂)₂— H 370 NH —(CH₂)₂S(O)₂NH(CH₂)₅— H 371 NH—(CH₂)₃(furan-2,5-ylene)(CH₂)₃— H 372 NH—(CH₂)₃(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 373 NH—(CH₂)₃(phen-1,3-ylene)(CH₂)₃— H 374 NH—(CH₂)₃(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 375 NH—(CH₂)₃(phen-1,4-ylene)(CH₂)₂— H 376 NH —(CH₂)₃(phen-1,4-ylene)(CH₂)₃— H377 NH —(CH₂)₃(phen-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 378 NH—(CH₂)₃(phen-1,4-ylene)NHC(O)(CH₂)₂— H 379 NH—(CH₂)₃(tetrahydrofuran-2,5-ylene)(CH₂)₃— H 380 NH—(CH₂)₃(thien-2,5-ylene)(CH₂)₃— H 381 NH—(CH₂)₃NH(phen-1,4-ylene)(CH₂)₂— H 382 NH —(CH₂)₃NHC(O)NH(CH₂)₅— H 383NH —(CH₂)₃O(phen-1,3-ylene)CH₂— H 384 NH —(CH₂)₃O(phen-1,4-ylene)(CH₂)₂—H 385 NH —(CH₂)₃O(phen-1,4-ylene)CH₂— H 386 NH—(CH₂)₄(phen-1,4-ylene)(CH₂)₂— H 387 NH —(CH₂)₄NH(phen-1,4-ylene)(CH₂)₂—H 388 NH —(CH₂)₅NH(phen-1,4-ylene)(CH₂)₂— H 389 NH —(CH₂)₇— H 390 NH—(CH₂)₈— H 391 NH 1-[—(CH₂)₂C(O)](piperidin-4-yl)(CH₂)₂— H 392 NH1-[—(CH₂)₂NHC(O)](piperidin-4-yl)- H 393 NH1-[—(CH₂)₂NHC(O)](piperidin-4-yl)(CH₂)₂— H 394 NH1-[—(CH₂)₃](piperidin-4-yl)CH₂— H 395 NH1-[—(CH₂)₃O(phen-1,4-ylene)(CH₂)₂](piperidin-4-yl)CH₂— H 396 NH1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)- H 397 NH1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₂— H 398 NH1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₃— H 399 NH1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)CH₂— H 400 NH1-[—CH₂(pyrid-2,6-ylene)CH₂](piperidin-4-yl)CH₂— H 401 NH2-[—(CH₂)₂](benzimidazol-5-yl)CH₂— H 402 NH2-[(S)—(—CH₂—](pyrrolidin-1-yl)C(O)(CH₂)₄— H 403 NH2-[(S)—(—CH₂—](pyrrolidin-1-yl)C(O)(phen-1,4-ylene)CH₂— H 404 NH4-[—(CH₂)₂—](piperidin-1-yl)(phen-1,4-ylene)(CH₂)₂— H 405 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₂— H 406 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₃— H 407 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₄— H 408 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₅— H 409 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(fur-2,5-ylene)CH₂— H 410 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂— H 411 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(thien-2,5-ylene)CH₂— H 412 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,2-ylene)CH₂— H 413 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,3-ylene)CH₂— H 414 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,4-ylene)CH₂— H 415 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)NH(phen-1,4-ylene)(CH₂)₂— H 416 NH4-[—CH₂—](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂— H 417 NH5-[—(CH₂)₂NHC(O)](pyrid-2-yl)CH₂— H 418 NH—CH₂(2-fluorophen-1,3-ylene)CH₂— H 419 NH—CH₂(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 420 NH—CH₂(phen-1,2-ylene)O(phen-1,2-ylene)CH₂— H 421 NH—CH₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— H 422 NH—CH₂(phen-1,4-ylene)NH(phen-1,4-ylene)CH₂— H 423 NH—CH₂CH(OH)CH₂NH(phen-1,4-ylene)(CH₂)₂— H 424 NH —(CH₂)₉— OH 425 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂— OH 426 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂CH₂— OH 427 NH—(CH₂)₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 428 NH—(CH₂)₂(phen-1,4-ylene)(CH₂)₂— OH 429 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂— OH 430 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂— OH 431 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂— OH 432 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂— OH 433 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,2-ylene)CH₂— OH 434 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,3-ylene)CH₂— OH 435 NH—(CH₂)₂(phen-1,4-ylene)NHC(O)CH₂O(phen-1,4-ylene)CH₂— OH 436 NH—(CH₂)₂-(trans-cyclohex-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 437 NH—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₂— OH 438 NH—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₄— OH 439 NH—(CH₂)₂-(trans-cyclohex-1,4-ylene)NHC(O)(CH₂)₅— OH 440 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(fur-2,5-ylene)CH₂— OH 441 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,3-ylene)CH₂— OH 442 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(phen-1,4-ylene)CH₂— OH 443 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)(thien-2,5-ylene)CH₂— OH 444 NH—(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,2- OH ylene)CH₂— 445NH —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,3- OH ylene)CH₂—446 NH —(CH₂)₂(trans-cyclohex-1,4-ylene)NHC(O)CH₂O(phen-1,4- OHylene)CH₂— 447 NH —(CH₂)₂C(O)N(CH₂CH₃)(phen-1,4-ylene)CH₂— OH 448 NH—(CH₂)₂C(O)NH(2-(CF₃O—)phen-1,4-ylene)CH₂— OH 449 NH—(CH₂)₂C(O)NH(2,3,5,6-tetrafluorophen-1,4-ylene)CH₂— OH 450 NH—(CH₂)₂C(O)NH(2,5-difluorophen-1,4-ylene)CH₂— OH 451 NH—(CH₂)₂C(O)NH(2,6-dichlorophen-1,4-ylene)CH₂— OH 452 NH—(CH₂)₂C(O)NH(2,6-diiodophen-1,4-ylene)CH₂— OH 453 NH—(CH₂)₂C(O)NH(2-bromophen-1,4-ylene)CH₂— OH 454 NH—(CH₂)₂C(O)NH(2-chloro-5-methoxyphen-1,4-ylene)CH₂— OH 455 NH—(CH₂)₂C(O)NH(2-chloro-6-methylphen-1,4-ylene)CH₂— OH 456 NH—(CH₂)₂C(O)NH(2-chlorophen-1,4-ylene)CH₂— OH 457 NH—(CH₂)₂C(O)NH(2-fluorophen-1,4-ylene)CH₂— OH 458 NH—(CH₂)₂C(O)NH(2-iodophen-1,4-ylene)CH₂— OH 459 NH—(CH₂)₂C(O)NH(2-methoxyphen-1,4-ylene)CH₂— OH 460 NH—(CH₂)₂C(O)NH(2-methylphen-1,4-ylene)CH₂— OH 461 NH—(CH₂)₂C(O)NH(3-chlorophen-1,4-ylene)CH₂— OH 462 NH—(CH₂)₂C(O)NH(4-chlorophen-1,3-ylene)CH₂— OH 463 NH—(CH₂)₂C(O)NH(4-methylphen-1,3-ylene)CH₂— OH 464 NH—(CH₂)₂C(O)NH(6-chlorophen-1,3-ylene)CH₂— OH 465 NH —(CH₂)₂C(O)NH(CH₂)₅—OH 466 NH —(CH₂)₂C(O)NH(CH₂)₆— OH 467 NH—(CH₂)₂C(O)NH(cis-cyclohex-1,4-ylene)CH₂— OH 468 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)(CH₂)₂NHC(O)CH₂— OH 469 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((S)-isomer) OH 470 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)C*H(CH₃)—((R)-isomer) OH 471 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)CH(CH₃)CH₂— OH 472 NH—(CH₂)₂C(O)NH(phen-1,4-ylene)CH₂NHC(O)CH₂— OH 473 NH—(CH₂)₂C(O)NH(trans-cyclohex-1,4-ylene)CH₂— OH 474 NH—(CH₂)₂C(O)NHCH₂(cyclohex-1,3-ylene)CH₂— OH 475 NH—(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)(CH₂)₂— OH 476 NH—(CH₂)₂C(O)NHCH₂(phen-1,3-ylene)CH₂— OH 477 NH—(CH₂)₂C(O)NHCH₂(phen-1,4-ylene)CH₂— OH 478 NH—(CH₂)₂C(O)NHCH₂(pyrid-2,6-ylene)CH₂— OH 479 NH—(CH₂)₂C(O)NHCH₂(trans-cyclohex-1,4-ylene)CH₂— OH 480 NH—(CH₂)₂N(CH₃)(CH₂)₂(cis-cyclohex-1,4-ylene)- OH 481 NH—(CH₂)₂N(CH₃)C(O)(3-nitrophen-1,4-ylene)CH₂— OH 482 NH—(CH₂)₂N(CH₃)C(O)(CH₂)₅— OH 483 NH —(CH₂)₂N(CH₃)C(O)(fur-2,5-ylene)CH₂—OH 484 NH —(CH₂)₂N(CH₃)C(O)(phen-1,3-ylene)CH₂— OH 485 NH—(CH₂)₂N(CH₃)C(O)(thien-2,5-ylene)CH₂— OH 486 NH—(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)- OH 487 NH—(CH₂)₂N(CH₃)C(O)(trans-cyclohex-1,4-ylene)CH₂— OH 488 NH—(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,3-ylene)CH₂— OH 489 NH—(CH₂)₂N(CH₃)C(O)CH₂O(phen-1,4-ylene)CH₂— OH 490 NH—(CH₂)₂N(CH₃)C(O)NH(phen-1,4-ylene)(CH₂)₂— OH 491 NH—(CH₂)₂N(CH₃)S(O)₂(phen-1,4-ylene)CH₂— OH 492 NH—(CH₂)₂NH(naphth-1,4-ylene)(CH₂)₂— OH 493 NH—(CH₂)₂NH(phen-1,4-ylene)(CH₂)₂— OH 494 NH —(CH₂)₂NHC(O)(CH₂)₅— OH 495NH —(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)- OH 496 NH—(CH₂)₂NHC(O)(cis-cyclopent-1,3-ylene)CH₂— OH 497 NH—(CH₂)₂NHC(O)(phen-1,4-ylene)(CH₂)₂— OH 498 NH—(CH₂)₂NHC(O)(phen-1,4-ylene)CH₂— OH 499 NH—(CH₂)₂NHC(O)(thien-2,5-ylene)CH₂— OH 500 NH—(CH₂)₂NHC(O)(trans-cyclohex-1,4-ylene)CH₂— OH 501 NH—(CH₂)₂NHC(O)NH(CH₂)₅— OH 502 NH —(CH₂)₂NHC(O)NH(phen-1,4-ylene)(CH₂)₂—OH 503 NH —(CH₂)₂NHC(O)NH(phen-1,4-ylene)CH₂— OH 504 NH—(CH₂)₂NHC(O)NHCH₂(phen-1,3-ylene)CH₂— OH 505 NH—(CH₂)₂NHC(O)NHCH₂(phen-1,4-ylene)CH₂— OH 506 NH—(CH₂)₂O(phen-1,2-ylene)O(CH₂)₂— OH 507 NH —(CH₂)₂O(phen-1,3-ylene)CH₂—OH 508 NH —(CH₂)₂O(phen-1,3-ylene)O(CH₂)₂— OH 509 NH—(CH₂)₂O(phen-1,4-ylene)C(O)(CH₂)₂— OH 510 NH—(CH₂)₂O(phen-1,4-ylene)O(CH₂)₂— OH 511 NH —(CH₂)₂S(O)₂NH(CH₂)₅— OH 512NH —(CH₂)₃(furan-2,5-ylene)(CH₂)₃— OH 513 NH—(CH₂)₃(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 514 NH—(CH₂)₃(phen-1,3-ylene)(CH₂)₃— OH 515 NH—(CH₂)₃(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 516 NH—(CH₂)₃(phen-1,4-ylene)(CH₂)₂— OH 517 NH —(CH₂)₃(phen-1,4-ylene)(CH₂)₃—OH 518 NH —(CH₂)₃(phen-1,4-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 519 NH—(CH₂)₃(phen-1,4-ylene)NHC(O)(CH₂)₂— OH 520 NH—(CH₂)₃(tetrahydrofuran-2,5-ylene)(CH₂)₃— OH 521 NH—(CH₂)₃(thien-2,5-ylene)(CH₂)₃— OH 522 NH—(CH₂)₃NH(phen-1,4-ylene)(CH₂)₂— OH 523 NH —(CH₂)₃NHC(O)NH(CH₂)₅— OH 524NH —(CH₂)₃O(phen-1,3-ylene)CH₂— OH 525 NH—(CH₂)₃O(phen-1,4-ylene)(CH₂)₂— OH 526 NH —(CH₂)₃O(phen-1,4-ylene)CH₂—OH 527 NH —(CH₂)₄(phen-1,4-ylene)(CH₂)₂— OH 528 NH—(CH₂)₄NH(phen-1,4-ylene)(CH₂)₂— OH 529 NH—(CH₂)₅NH(phen-1,4-ylene)(CH₂)₂— OH 530 NH —(CH₂)₇— OH 531 NH —(CH₂)₈—OH 532 NH 1-[—(CH₂)₂C(O)](piperidin-4-yl)(CH₂)₂— OH 533 NH1-[—(CH₂)₂NHC(O)](piperidin-4-yl)- OH 534 NH1-[—(CH₂)₂NHC(O)](piperidin-4-yl)(CH₂)₂— OH 535 NH1-[—(CH₂)₃](piperidin-4-yl)CH₂— OH 536 NH1-[—(CH₂)₃O(phen-1,4-ylene)(CH₂)₂](piperidin-4-yl)CH₂— OH 537 NH1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)- OH 538 NH1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₂— OH 539 NH1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)(CH₂)₃— OH 540 NH1-[—CH₂(2-fluorophen-1,3-ylene)CH₂](piperidin-4-yl)CH₂— OH 541 NH1-[—CH₂(pyrid-2,6-ylene)CH₂](piperidin-4-yl)CH₂— OH 542 NH2-[—(CH₂)₂](benzimidazol-5-yl)CH₂— OH 543 NH2-[(S)—(—CH₂—](pyrrolidin-1-yl)C(O)(CH₂)₄— OH 544 NH2-[(S)—(—CH₂—](pyrrolidin-1-yl)C(O)(phen-1,4-ylene)CH₂— OH 545 NH4-[—(CH₂)₂—](piperidin-1-yl)(phen-1,4-ylene)(CH₂)₂— OH 546 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₂— OH 547 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₃— OH 548 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₄— OH 549 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(CH₂)₅— OH 550 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(fur-2,5-ylene)CH₂— OH 551 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂— OH 552 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)(thien-2,5-ylene)CH₂— OH 553 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,2-ylene)CH₂— OH 554 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,3-ylene)CH₂— OH 555 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)CH₂O(phen-1,4-ylene)CH₂— OH 556 NH4-[—(CH₂)₂](piperidin-1-yl)C(O)NH(phen-1,4-ylene)(CH₂)₂— OH 557 NH4-[—CH₂—](piperidin-1-yl)C(O)(phen-1,4-ylene)CH₂— OH 558 NH5-[—(CH₂)₂NHC(O)](pyrid-2-yl)CH₂— OH 559 NH—CH₂(2-fluorophen-1,3-ylene)CH₂— OH 560 NH—CH₂(phen-1,2-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 561 NH—CH₂(phen-1,2-ylene)O(phen-1,2-ylene)CH₂— OH 562 NH—CH₂(phen-1,3-ylene)NH(phen-1,4-ylene)(CH₂)₂— OH 563 NH—CH₂(phen-1,4-ylene)NH(phen-1,4-ylene)CH₂— OH 564 NH—CH₂CH(OH)CH₂NH(phen-1,4-ylene)(CH₂)₂— OH

Another group of representative compounds of this invention arecompounds of formula If:

wherein W, R^(1A), R^(1B), R^(1C), R^(2A), R^(2B), R⁴ and R⁶ are asdefined in Table II; or a pharmaceutically acceptable salt or solvatethereof.

TABLE II Ex. W R^(1A) R^(1B) R^(1C) R^(2A) R^(2B) R⁴ R⁶ 565 O H H H Br H—(CH₂)₉— H 566 O F H H H H —(CH₂)₉— H 567 O H Cl H F F —(CH₂)₉— H 568 OH Cl Cl F F —(CH₂)₉— H 569 O H H H F F —(CH₂)₉— H 570 O H H H Br H—(CH₂)₂C(O)NH(phen-1,4- H ylene)CH₂— 571 O F H H H H—(CH₂)₂C(O)NH(phen-1,4- H ylene)CH₂— 572 O H Cl H F F—(CH₂)₂C(O)NH(phen-1,4- H ylene)CH₂— 573 O H Cl Cl F F—(CH₂)₂C(O)NH(phen-1,4- H ylene)CH₂— 574 O H H H F F—(CH₂)₂C(O)NH(phen-1,4- H ylene)CH₂— 575 O H H H Br H —(CH₂)₉— OH 576 OF H H H H —(CH₂)₉— OH 577 O H Cl H F F —(CH₂)₉— OH 578 O H Cl Cl F F—(CH₂)₉— OH 579 O H H H F F —(CH₂)₉— OH 580 O H H H Br H—(CH₂)₂C(O)NH(phen-1,4- OH ylene)CH₂— 581 O F H H H H—(CH₂)₂C(O)NH(phen-1,4- OH ylene)CH₂— 582 O H Cl H F F—(CH₂)₂C(O)NH(phen-1,4- OH ylene)CH₂— 583 O H Cl Cl F F—(CH₂)₂C(O)NH(phen-1,4- OH ylene)CH₂— 584 O H H H F F—(CH₂)₂C(O)NH(phen-1,4- OH ylene)CH₂—Definitions

When describing the compounds, compositions, methods and processes ofthis invention, the following terms have the following meanings unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

The term “alkylene” means a divalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “alkoxy” means a monovalent group of the formula (alkyl)-O—,where alkyl is as defined herein. Representative alkoxy groups include,by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,sec-butoxy, isobutoxy, tert-butoxy and the like.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbonatoms. Representative alkenyl groups include, by way of example,ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and thelike. The term “alkenylene” means a divalent alkenyl group.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbonatoms. Representative alkynyl groups include, by way of example,ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like. The term“alkynylene” means a divalent alkynyl group.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms. Representative aryl groups include, by way of example, phenyl andnaphthalene-1-yl, naphthalene-2-yl, and the like. The term “arylene”means a divalent aryl group.

The term “azacycloalkyl” means a monovalent heterocyclic ring containingone nitrogen atom, i.e., a cycloalkyl group in which one carbon atom hasbeen replaced with a nitrogen atom. Unless otherwise defined, suchazacycloalkyl groups typically contain from 2 to 9 carbon atoms.Representative examples of an azacycloalkyl group are pyrrolidinyl andpiperidinyl groups. The term “azacycloalkylene” means a divalentazacycloakyl group. Representative examples of an azacycloalkylene groupare pyrrolidinylene and piperidinylene groups.

The term “azabicycloalkyl” means a monovalent heterobicyclic ringcontaining one nitrogen atom, i.e., a bicycloalkyl group in which onecarbon atom has been replaced with a nitrogen atom. Unless otherwisedefined, such azabicycloalkyl groups typically contain from 5 to 10carbon atoms. Representative azabicycloalkyl groups include2-azabicyclo[2.2.1]heptyl, 7-azabicyclo[2.2.1]heptyl,1-azabicyclo[2.2.2]octyl, 2-azabicyclo[2.2.2]octyl,9-azabicyclo[4.2.1]nonyl, 3-azabicyclo[3.3.2]decyl,9-azabicyclo[3.3.2]decyl and the like. The term “azabicycloalkylene”means a divalent azabicycloakyl group.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms. Representative cycloalkylgroups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and the like. The term “cycloalkylene” means a divalentcycloalkyl group.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms. Representative heteroaryl groupsinclude, by way of example, monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like, wherethe point of attachment is at any available carbon or nitrogen ringatom. The term “heteroarylene” means a divalent heteroaryl group.

The term “heterocyclyl” or “heterocyclic” means a monovalent saturatedor unsaturated (non-aromatic) group having a single ring or multiplecondensed rings and containing in the ring at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen or sulfur.Unless otherwise defined, such heterocyclic groups typically containfrom 2 to 9 total ring carbon atoms. Representative heterocyclic groupsinclude, by way of example, monovalent species of pyrrolidine,imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine,thiomorpholine, piperazine, 3-pyrroline and the like, where the point ofattachment is at any available carbon or nitrogen ring atom. The term“heterocyclene” means a divalent heterocyclyl or heterocyclic group.

which is substituted and optionally substituted as defined herein.

The term “divalent hydrocarbon group” means a divalent hydrocarbon groupwhich is composed primarily of carbon and hydrogen atoms and whichoptionally contains one or more heteroatoms. Such divalent hydrocarbongroups may be branched or unbranched, saturated or unsaturated, acyclicor cyclic, aliphatic or aromatic, or combinations thereof. The divalenthydrocarbon group can optionally contain heteroatoms incorporated intothe hydrocarbon chain or as substituents attached to the hydrocarbonchain.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown in parenthesespreceding the term. For example, the term “(1-4C)alkyl” means an alkylgroup having from 1 to 4 carbon atoms.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. Salts derived from pharmaceutically acceptable inorganic basesinclude ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,manganic, manganous, potassium, sodium, zinc and the like. Particularlypreferred are ammonium, calcium, magnesium, potassium and sodium salts.Salts derived from pharmaceutically acceptable organic bases includesalts of primary, secondary and tertiary amines, including substitutedamines, cyclic amines, naturally-occurring amines and the like, such asarginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable acids include acetic, ascorbic,benzenesulfonic, benzoic, camphosulfonic, citric, ethanesulfonic,edisylic, fumaric, gentisic, gluconic, glucoronic, glutamic, hippuric,hydrobromic, hydrochloric, isethionic, lactic, lactobionic, maleic,malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic, nicotinic,nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric,tartaric, p-toluenesulfonic, xinafoic and the like. Particularlypreferred are citric, hydrobromic, hydrochloric, isethionic, maleic,naphthalene-1,5-disulfonic, phosphoric, sulfuric and tartaric acids.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. Preferably, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of intermediatecompounds that are not intended for administration to a patient.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically acceptablesalt or solvate or stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically acceptable salt of a stereoisomer of a compound ofthis invention.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patient,such as a mammal (particularly a human) that includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitutionreaction, such as a nucleophilic substitution reaction. By way ofexample, representative leaving groups include chloro, bromo and iodogroups; sulfonic ester groups, such as mesylate, tosylate, brosylate,nosylate and the like; and acyloxy groups, such as acetoxy,trifluoroacetoxy and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups that may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, New York, 1999, and references cited therein.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “carboxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxy group. Representativecarboxy-protecting groups include, but are not limited to, esters, suchas methyl, ethyl, tert-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluoroenylmethyl (Fm), trimethylsilyl (TMS), tert-butyldimethylsilyl(TBS), diphenylmethyl (benzhydryl, DPM) and the like.

The term “hydroxyl-protecting group” means a protecting group suitablefor preventing undesirable reactions at a hydroxyl group. Representativehydroxyl-protecting groups include, but are not limited to, silyl groupsincluding tri(1-6C)alkylsilyl groups, such as trimethylsilyl (TMS),triethylsilyl (TES), tert-butyldimethylsilyl (TBS) and the like; esters(acyl groups) including (1-6C)alkanoyl groups, such as formyl, acetyland the like; arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl(PMB), 9-fluorenylmethyl (Fm), diphenylmethyl (benzhydryl, DPM) and thelike. Additionally, two hydroxyl groups can also be protected as analkylidene group, such as prop-2-ylidine, formed, for example, byreaction with a ketone, such as acetone.

General Synthetic Procedures

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures orby using other information readily available to those of ordinary skillin the art. Although a particular embodiment of the present inventionmay be shown or described herein, those skilled in the art willrecognize that all embodiments or aspects of the present invention canbe prepared using the methods described herein or by using othermethods, reagents and starting materials known to those skilled in theart. It will also be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. While the optimumreaction conditions may vary depending on the particular reactants orsolvent used, such conditions can be readily determined by one ofordinary skill in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions for protection and deprotection of suchfunctional groups are well-known in the art. Protecting groups otherthan those illustrated in the procedures described herein may be used,if desired. For example, numerous protecting groups, and theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York,1999, and references cited therein.

By way of illustration, the compounds of this invention can be preparedby a process comprising:

(a) reacting a compound of formula 1:

or a salt thereof; with a compound of formula 2:

wherein X¹ represents a leaving group, R^(6a) represents hydrogen orOP¹, and P¹ and P² each independently represent a hydrogen atom or ahydroxyl-protecting group;

(b) reacting a compound of formula 3:

or salt thereof; with a compound of formula 4:

wherein X² represents a leaving group, R^(6b) represents hydrogen orOP³, and P³ and P⁴ each independently represent a hydrogen atom or ahydroxyl-protecting group;

(c) coupling a compound of formula 5:

with a compound of formula 6:

wherein X^(Qa) and X^(Qb) each independently represent functional groupsthat couple to form a group Q, P^(5a) represents a hydrogen atom or anamino-protecting group, R^(6c) represents hydrogen or OP^(5b), andP^(5b) and P⁶ each independently represent a hydrogen atom or ahydroxyl-protecting group;

(d) for a compound of formula I wherein R⁵ represents a hydrogen atom,reacting a compound of formula 3 with a compound of formula 7a or 7b:

or a hydrate thereof (e.g., a glyoxal), in the presence of a reducingagent, wherein P⁷ represents a hydrogen atom or a hydroxyl-protectinggroup;

(e) reacting a compound of formula 1 with a compound of formula 8:

or a hydrate thereof, in the presence of a reducing agent, whereinR^(6d) represents hydrogen or OP⁸, P⁸ and P⁹ each independentlyrepresent a hydrogen atom or a hydroxyl-protecting group, P¹⁰ representsa hydrogen atom or an amino-protecting group, and R^(4′) represents aresidue that, together with the carbon to which it is attached, affordsa group R⁴ upon completion of the reaction;

(f) reacting a compound of formula 9:

wherein X³ represents a leaving group, with a compound of formula 10:

wherein R^(6e) represents hydrogen or OP¹¹, P¹¹ and P¹² eachindependently represent a hydrogen atom or a hydroxyl-protecting group,and P¹³ represents a hydrogen atom or an amino-protecting group; or

(g) reacting a compound of formula 11:

or a hydrate thereof; wherein R^(4′) represents a residue that, togetherwith the carbon to which it is attached, affords a group R⁴ uponcompletion of the reaction; with a compound of formula 10 in thepresence of a reducing agent; and then

removing any protecting group P¹, P², P³, P⁴, P^(5a), P^(5b), P⁶, P⁷,P⁸, P⁹, P¹⁰, P¹¹, P¹² or P¹³ to provide a compound of formula I; andoptionally, forming a pharmaceutically acceptable salt thereof.

Generally, if a salt of one of the starting materials is used in theprocesses described above, such as an acid addition salt, the salt istypically neutralized before or during the reaction process. Thisneutralization reaction is typically accomplished by contacting the saltwith one molar equivalent of a base for each molar equivalent of acidaddition salt.

In process (a), i.e., the reaction between the compounds of formula 1and 2, the leaving group represented by X¹ can be, for example, halo,such as chloro, bromo or iodo, or a sulfonic ester group, such asmesylate or tosylate. The groups P¹ and P² can be, for example,trimethylsilyl and benzyl or methyl, respectively. This reaction istypically conducted in an inert diluent, such as acetonitrile, in thepresence of a base. For example, this reaction can be conducted in thepresence of a tertiary amine, such as diisopropylethylamine. Generally,this reaction is conducted at a temperature in the range of from 0° C.to 100° C. until the reaction is substantially complete. The reactionproduct is then isolated using conventional procedures, such asextraction, recrystallization, chromatography and the like.

Compounds of formula 1 are generally known in the art or can be preparedfrom commercially available starting materials and reagents usingwell-known procedures. For example, compounds of formula 1 can beprepared by deprotecting a compound of formula 12:

wherein P¹⁴ represents an amino-protecting group, such as a benzylgroup. By way of illustration, a benzyl group can be readily removed byreduction using, for example, hydrogen or ammonium formate and a groupVIII metal catalyst, such as palladium on carbon. When W representsNW^(a), the hydrogenation reaction is conveniently performed usingPearlman's catalyst (i.e., Pd(OH)₂).

Compounds of formula 12 can be prepared by reacting an isocyanatecompound of formula 13:

with a compound of formula 14:

Compounds of formula 2 can be prepared by various procedures describedherein or by procedures that are well-known to those skilled in the art.In this regard, the preparation of related compounds is described inU.S. Pat. Nos. 5,648,370; 5,763,465; 5,846,989; 5,929,100; 5,973,167;5,977,384; 6,008,365; and 6,080,869; in International Patent PublicationNos. WO 92/08708; WO 93/23385; WO 93/24473; WO 97/10227; WO 97/23470; WO99/09018; WO 00/50413 and WO 2004/016601 A1; and in Weistock et al, J.Med. Chem. 1987, 30, 1166-1176. By way of illustration, the hydroxylgroup of a compound of formula 23 below, can be readily converted into aleaving group using well-known reagents and procedures. For example, ahydroxyl group can be converted into a halo group using an inorganicacid halide, such as thionyl chloride, phosphorous trichloride,phosphorous tribromide, phosphorous oxychloride and the like, or ahalogen acid, such a hydrogen bromide.

In process (b), i.e., the reaction of a compound of formula 3 with acompound of formula 4, the leaving represented by X² can be, forexample, halo, such as chloro, bromo or iodo, or a sulfonic ester group,such as mesylate or tosylate. The groups P³ and P⁴ can be, for example,tert-butyldimethylsilyl and benzyl or methyl, respectively. Thisreaction is typically conducted in the presence of a base, such assodium bicarbonate, and an alkali metal iodide, such as sodium iodide.Generally, this reaction is conducted in an inert diluent, such astetrahydrofuran, at a temperature ranging from 25° C. to 100° C. untilthe reaction is substantially complete. The reaction product is thenisolated using conventional procedures, such as extraction,recrystallization, chromatography and the like.

Compounds of formula 3 can be prepared by deprotecting a compound offormula 15:

wherein one or both of P¹⁵ and P¹⁶ independently represents a protectinggroup, such as tert-butoxycarbonyl, and any remainder represents ahydrogen atom. For example, a tert-butoxycarbonyl group can be removedby treating the protected compound with trifluoroacetic acid.

Compounds of formula 15 can be prepared by reacting a compound offormula 1 with a compound of formula 16:X³—R⁴—NP¹⁵P¹⁶  16

wherein X³ represents a leaving group such as halo, such as chloro,bromo or iodo, or sulfonic ester group, such as mesylate or tosylate.This reaction is typically conducted by contacting a compound of formula1 with a compound of formula 16 in an inert diluent, such asacetonitrile, DMF or mixtures thereof, at a temperature ranging fromabout 0° C. to about 100° C. until the reaction is substantiallycomplete.

Alternatively, compounds of formula 3 can be obtained by reductiveamination of a compound of formula 11. The reductive amination can beperformed by reacting the compound of formula 11 with, for example,benzylamine and hydrogen in the presence of palladium on carbon.

Compounds of formula 11 may be prepared by oxidizing the correspondingalcohol of formula 17:

using a suitable oxidizing agent, such as sulfur trioxide pyridinecomplex and dimethyl sulfoxide. This oxidation reaction is typicallyconducted in an inert diluent, such as dichloromethane, the presence ofa tertiary amine, such as diisopropylethylamine, at a temperatureranging from about −20° C. to about 25° C.

Compounds of formula 17 can be prepared by reacting a compound offormula 1 with a compound of formula 18:X⁴—R⁴—OH   18wherein X⁴ represents a leaving group such as halo, such as chloro,bromo or iodo, or a sulfonic ester group, such as mesylate or tosylate.

Compounds of formula 4 in which R^(6b) represents OP³ can be prepared byreacting a compound of formula 19:

with a reducing agent, such as borane, and then protecting the resultinghydroxyl group if necessary. If desired, such a reduction can beperformed in the presence of a chiral catalyst to provide compounds offormula 4 in chiral form. For example, compounds of formula 19 can bereduced in the presence of a chiral catalyst formed from(R)-(+)-α,α-diphenyl-2-pyrrolidinemethanol and trimethylboroxine; oralternatively, from (S)-(−)-α,α-diphenyl-2-pyrrolidinemethanol andtrimethylboroxine. The resulting hydroxyl group can then be protectedwith a hydroxyl-protecting group, P³, by reaction with, for example,tert-butyldimethylsilyl trifluoromethanesulfonate.

Compounds of formula 19 in which X² represents a bromine atom can beprepared by reacting a compound of formula 20:

with bromine in the presence of a Lewis acid, such as boron trifluoridediethyl etherate. Compounds of formula 20 are well-known in the art orcan be prepared by well-known procedures using commercially availablestarting materials and reagents.

Referring to process (c), i.e., the reaction of a compound of formula 5with a compound of formula 6, it will be appreciated that the groupsX^(Qa) and X^(Qb) should be selected so as to afford the desired group Qupon completion of the reaction. For example, when the desired group Qis an amide group, i.e., —N(Q^(a))C(O)— or —C(O)N(Q^(b)), one of X^(Qa)and X^(Qb) can be an amine group (i.e., —NHQ^(a) or —NHQ^(b)) and theother can be a carboxyl group (i.e., —COOH) or a reactive derivativethereof (such as acyl halide, such as an acyl chloride or acyl bromide).The groups P^(5a), P^(5b) and P⁶ can be, for example, benzyl,trimethylsilyl and benzyl or methyl, respectively. When Q is an amidegroup, the reaction can be performed under conventional amide couplingconditions. Similarly, when the desired group Q is a sulfonamide, i.e.,—N(Q^(c))S(O)₂— or —S(O)₂N(Q^(d))-, one of X^(Qa) and X^(Qb) can be anamine group, —NHQ^(c) or —NHQ^(d) and the other can be a sulfonyl halidegroup (such as sulfonyl chloride or sulfonyl bromide).

Compounds of formula 5 can be prepared by reacting a compound of formula1 with a compound of formula 21:X⁵—(R^(4a))_(d)-(A¹)_(e)-(R_(4b))_(f)X^(Qa′)  21wherein X⁵ represents a leaving group including halo, such as chloro,bromo or iodo, and a sulfonic ester group, such as mesylate or tosylate;and X^(Qa′) represents X^(Qa), such as a carboxyl group or an aminogroup NHQ^(a), or a protected derivative thereof, such as a(1-6C)alkoxycarbonylamino group or a tert-butoxycarbonylamino group.This reaction is typically conducted by a method analogous to that usedto prepare compounds of formula 3, followed by removing any protectinggroup in X^(Qa′).

Compounds of formula 6 can be prepared by reacting a compound of formula10 (where P^(5a)=P¹³, P⁶=P¹², and R^(6e)=R^(6e)) with a compound offormula 22:X^(Qb′)-(R^(4c))_(g)-(A²)_(h)-(R^(d))_(i)—X⁶  22wherein X⁶ represents a leaving group including halo, such as chloro,bromo or iodo, and a sulfonic ester group, such as mesylate or tosylate;and X^(Qb′) represents X^(Qb), such as a carboxyl group or an aminogroup NHQ^(b), or a protected derivative thereof, such as a(1-6C)alkoxycarbonyl group or a tert-butoxycarbonylamino group. Thisreaction is typically conducted by a method analogous to that used toprepare compounds of formula 3, followed by removing any protectinggroup in X^(Qb′).

Referring to process (d), i.e., the reaction of a compound of formula 3with a compound of formula 7a or 7b, any suitable reducing agent may beused in this reaction. For example, the reducing agent can be hydrogenin the presence of a Group VIII metal catalyst, such as palladium oncarbon; or a metal hydride reagent, such as sodiumtriacetoxyborohydride. The group P⁷ can be, for example, benzyl ormethyl. This reaction is typically conducted in an inert diluent and aprotic solvent, such as a mixture of dichloroethane and methanol, at atemperature in the range of from 0° C. to 100° C. until the reaction issubstantially complete.

Compounds of formula 7a or 7b in the form of a hydrate can be preparedby conventional procedures. For example, a compound of formula 7a can beprepared by dibrominating a compound of formula 20 and then hydrolyzingthe resulting dibromide to form a glyoxal or a hydrate thereof. Forexample, a compound of formula 20 can be reacted with hydrogen bromideand then hydrolyzed with water to form the corresponding glyoxalhydrate. Compounds of formula 7b can be prepared, for example, byoxidation of the corresponding alcohol or reduction of the correspondingnitrile or carboxylic acid or ester using conventional reagents andprocedures.

Referring to process (e), i.e., the reaction of a compound of formula 1with a compound of formula 8, any suitable reducing agent may be used inthis reaction. For example, the reducing agent may be hydrogen in thepresence of a Group VIII metal catalyst, such as palladium on carbon; ora metal hydride reagent, such as sodium triacetoxyborohydride. Thegroups P⁸, P⁹ and P¹⁰ can be, for example, trimethylsilyl, benzyl ormethyl and benzyl, respectively. Typically, this reduction reaction isconducted in an inert diluent and a protic solvent, such asdichloroethane and methanol, at a temperature in the range of from 0° C.to 100° C. until the reaction is substantially complete.

Compounds of formula 8 may be prepared by oxidizing a compound offormula 23:

using any suitable oxidizing agent, such as sulfur trioxide pyridinecomplex and dimethyl sulfoxide. This reaction is typically conducted inthe presence of a tertiary amine, such as diisopropylethylamine, at atemperature in the range of from about −20° C. to about 25° C. until theoxidation is substantially complete.

Compounds of formula 23 can be prepared by reacting a compound offormula 10 (where P¹⁰=P¹³, P⁹=P¹², and R^(6d)=R^(6e)) with a compound offormula 24:HO—R⁴—X⁷  24wherein X⁷ represents a leaving group including halo, such as chloro,bromo or iodo, and a sulfonic ester group, such as mesylate or tosylate.

Referring to process (f), i.e., the reaction of a compound of formula 9with a compound of formula 10, the leaving group represented by X³ canbe, for example, halo, such as chloro, bromo or iodo, or a sulfonicester group, such as mesylate or tosylate. The groups P¹¹, P¹² and P¹³can be, for example, trimethylsilyl, benzyl or methyl, and benzyl,respectively. This reaction is typically conducted an inert diluent,such as acetonitrile, in the presence of a suitable base. For example,this reaction can be conducted in the presence of a tertiary amine, suchas diisopropylethylamine. Generally, this reaction is conducted at atemperature in the range of from 0° C. to 100° C. until the reaction issubstantially complete.

Compounds of formula 9 can be prepared by steps analogous to those ofmethods (a) to (e) herein, starting from a compound of formula 1.Additionally, compounds of formula 10 can be prepared from compounds offormula 4 by reaction with an amine of formula P¹³NH₂.

Referring to process (g), i.e., the reaction of a compound of formula 11with a compound of formula 10, any suitable reducing agent may be usedin this reaction. For example, the reducing agent may be hydrogen in thepresence of a Group VIII metal catalyst, such as palladium on carbon; ora metal hydride reagent, such as sodium triacetoxyborohydride. Thegroups P¹¹, P¹² and P¹³ can be, for example, tert-butyldimethylsilyl,benzyl and benzyl, respectively. Typically, this reduction reaction isconducted in an inert diluent and a protic solvent, such asdichloroethane and methanol, at a temperature in the range of from 0° C.to 100° C. until the reaction is substantially complete.

Compounds of formula 11 are readily prepared by oxidation of thecorresponding alcohol or by hydrolysis of the corresponding acetal. Anysuitable oxidizing agent may be employed in this reaction to provide thealdehyde, such as sulfur trioxide pyridine complex and dimethylsulfoxide. The acetal may be hydrolyzed under conventional conditionsusing aqueous acid to provide the aldehyde.

In a particular embodiment, certain compounds of formula I are preparedby a process comprising:

(h) contacting a compound of formula 25:

with a reagent selected from boron tribromide, boron trichloride,hydrobromic acid and hydrochloric acid to form a compound of formula Ior a salt or stereoisomer thereof; and, optionally, forming apharmaceutically acceptable salt of the compound of formula I.

Referring to process (h), this reaction is typically conducted bycontacting the compound of formula 25 with an excess, such as about 2 toabout 6 molar equivalents, of boron tribromide at a temperature rangingfrom about −30° C. to about 30° C. for about 1 to about 24 hours oruntil the reaction is substantially complete. The reaction product isthen isolated using conventional procedures, such as extraction,recrystallization, chromatography and the like. Compounds of formula 25can be prepared by the methods described herein, such as by processes(a) to (g).

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of this invention orintermediates thereof are described in the Examples set forth below.

Pharmaceutical Compositions and Formulations

The compounds of this invention are typically administered to a patientin the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to,inhaled, oral, nasal, topical (including transdermal) and parenteralmodes of administration.

It will be understood that any form of the compounds of this invention,(i.e., free base, pharmaceutically acceptable salt, solvate, etc.) thatis suitable for the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one of its compositions aspects, this invention isdirected to a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or excipient and a therapeutically effective amountof a compound of this invention, or a pharmaceutically acceptable saltthereof. Optionally, such pharmaceutical compositions may contain othertherapeutic and/or formulating agents if desired.

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a compound of the present inventionor a pharmaceutically acceptable salt thereof. Typically, suchpharmaceutical compositions will contain from about 0.01 to about 95% byweight of the active agent; including, from about 0.01 to about 30% byweight; such as from about 0.01 to about 10% by weight of the activeagent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;(21) compressed propellant gases, such as chlorofluorocarbons andhydrofluorocarbons; and (22) other non-toxic compatible substancesemployed in pharmaceutical compositions.

The pharmaceutical compositions of this invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills, canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of this invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices, such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a nebulizer inhaler. Such nebulizer devices typically produce astream of high velocity air that causes the pharmaceutical compositioncomprising the active agent to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the active agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having about 90% or more of the particles with a diameter ofless than about 10 μm. Suitable nebulizer devices are providedcommercially, for example, by PARI GmbH (Starnberg, German). Othernebulizer devices include Respimat (Boehringer Ingelheim) and thosedisclosed, for example, in U.S. Pat. No. 6,123,068 and WO 97/12687.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an isotonic aqueous solution comprising from about0.05 μg/mL to about 10 mg/mL of a compound of this invention or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

In another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a dry powder inhaler. Such dry powder inhalers typicallyadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. In order to achieve a freeflowing powder, the active agent is typically formulated with a suitableexcipient such as lactose or starch.

A representative pharmaceutical composition for use in a dry powderinhaler comprises dry lactose having a particle size between about 1 μmand about 100 μm and micronized particles of a compound of thisinvention, or a pharmaceutically acceptable salt or solvate orstereoisomer thereof.

Such a dry powder formulation can be made, for example, by combining thelactose with the active agent and then dry blending the components.Alternatively, if desired, the active agent can be formulated without anexcipient. The pharmaceutical composition is then typically loaded intoa dry powder dispenser, or into inhalation cartridges or capsules foruse with a dry powder delivery device.

Examples of dry powder inhaler delivery devices include Diskhaler(GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Pat.No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No.6,378,519; Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S.Pat. No. 4,524,769); Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat.No. 4,353,365) and Handihaler (Boehringer Ingelheim). Further examplesof suitable DPI devices are described in U.S. Pat. Nos. 5,415,162,5,239,993, and 5,715,810 and references cited therein.

In yet another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a metered-dose inhaler. Such metered-dose inhalers typicallydischarge a measured amount of the active agent or a pharmaceuticallyacceptable salt thereof using compressed propellant gas. Accordingly,pharmaceutical compositions administered using a metered-dose inhalertypically comprise a solution or suspension of the active agent in aliquefied propellant. Any suitable liquefied propellant may be employedincluding chlorofluorocarbons, such as CCl₃F, and hydrofluoroalkanes(HFAs), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Due to concerns aboutchlorofluorocarbons affecting the ozone layer, formulations containingHFAs are generally preferred. Additional optional components of HFAformulations include co-solvents, such as ethanol or pentane, andsurfactants, such as sorbitan trioleate, oleic acid, lecithin, andglycerin. See, for example, U.S. Pat. No. 5,225,183, EP 0717987 A2, andWO 92/22286.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a compoundof this invention, or a pharmaceutically acceptable salt or solvate orstereoisomer thereof; from about 0% to about 20% by weight ethanol; andfrom about 0% to about 5% by weight surfactant; with the remainder beingan HFA propellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing theactive agent, ethanol (if present) and the surfactant (if present). Toprepare a suspension, the active agent is micronized and then combinedwith the propellant. The formulation is then loaded into an aerosolcanister, which forms a portion of a metered-dose inhaler device.Examples of metered-dose inhaler devices developed specifically for usewith HFA propellants are provided in U.S. Pat. Nos. 6,006,745 and6,143,277. Alternatively, a suspension formulation can be prepared byspray drying a coating of surfactant on micronized particles of theactive agent. See, for example, WO 99/53901 and WO 00/61108.

For additional examples of processes of preparing respirable particles,and formulations and devices suitable for inhalation dosing see U.S.Pat. Nos. 6,268,533, 5,983,956, 5,874,063, and 6,221,398, and WO99/55319 and WO 00/30614.

In another embodiment, the pharmaceutical compositions of this inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof this invention will typically comprise a compound of the presentinvention as the active ingredient and one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate.Optionally or alternatively, such solid dosage forms may also comprise:(1) fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8)absorbents, such as kaolin and/or bentonite clay; (9) lubricants, suchas talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and/or mixtures thereof; (10) coloringagents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of this invention. Examples ofpharmaceutically acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof this invention are preferably packaged in a unit dosage form. Theterm “unit dosage form” means a physically discrete unit suitable fordosing a patient, i.e., each unit containing a predetermined quantity ofactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

The compounds of this invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example, acompound of this invention can be admixed with permeation enhancers,such as propylene glycol, polyethylene glycolm monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The pharmaceutical compositions of this invention may also contain othertherapeutic agents that are co-administered with a compound of thisinvention, or pharmaceutically acceptable salt or solvate orstereoisomer thereof. For example, the pharmaceutical compositions ofthis invention may further comprise one or more therapeutic agentsselected from other bronchodilators (e.g., PDE₃ inhibitors, adenosine 2bmodulators and β₂ adrenergic receptor agonists); anti-inflammatoryagents (e.g. steroidal anti-inflammatory agents, such ascorticosteroids; non-steroidal anti-inflammatory agents (NSAIDs), andPDE₄ inhibitors); other muscarinic receptor antagonists (i.e.,anticholinergic agents); antiinfective agents (e.g. Gram positive andGram negative antibiotics or antivirals); antihistamines; proteaseinhibitors; and afferent blockers (e.g., D₂ agonists and neurokininmodulators). The other therapeutic agents can be used in the form ofpharmaceutically acceptable salts or solvates. Additionally, ifappropriate, the other therapeutic agents can be used as optically purestereoisomers.

Representative β₂ adrenergic receptor agonists that can be used incombination with, and in addition to, the compounds of this inventioninclude, but are not limited to, salmeterol, salbutamol, formoterol,salmefamol, fenoterol, terbutaline, albuterol, isoetharine,metaproterenol, bitolterol, pirbuterol, levalbuterol and the like, orpharmaceutically acceptable salts thereof. Other β₂ adrenergic receptoragonists that can be used in combination with the compounds of thisinvention include, but are not limited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422, published on Aug. 29,2002;3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490, published Sep. 12,2002;3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamideand related compounds disclosed in WO 02/076933, published on Oct. 3,2002;4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds disclosed in WO 03/024439, published on Mar. 27,2003; and pharmaceutically acceptable salts thereof. When employed, theβ₂-adrenoreceptor agonist will be present in the pharmaceuticalcomposition in a therapeutically effective amount. Typically, theβ₂-adrenoreceptor agonist will be present in an amount sufficient toprovide from about 0.05 μg to about 500 μg per dose.

Representative steroidal anti-inflammatory agents that can be used incombination with the compounds of this invention include, but are notlimited to, methyl prednisolone, prednisolone, dexamethasone,fluticasone propionate,6,9-difluoro-17-[(2-furanylcarbonyl)oxy]-11-hydroxy-16-methyl-3-oxoandrosta-1,4-diene-17-carbothioicacid S-fluoromethyl ester,6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-propionyloxy-androsta-1,4-diene-17-carbothioicacid S-(2-oxo-tetrahydrofuran-3S-yl) ester, beclomethasone esters (e.g.the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g. the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,ST-126 and the like, or pharmaceutically-acceptable salts thereof. Whenemployed, the steroidal anti-inflammatory agent will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the steroidal anti-inflammatory agent will be present in anamount sufficient to provide from about 0.05 μg to about 500 μg perdose.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (such as sodium cromoglycate;nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g.theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g. monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors, such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g. adenosine 2a agonists); cytokineantagonists (e.g. chemokine antagonists such as, an interleukin antibody(IL antibody), specifically, an IL-4 therapy, an IL-13 therapy, or acombination thereof); or inhibitors of cytokine synthesis.

For example, representative phosphodiesterase-4 (PDE4) inhibitors ormixed PDE3/PDE4 inhibitors that can be used in combination with thecompounds of this invention include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents)that can be used in combination with, and in addition to, the compoundsof this invention include, but are not limited to, atropine, atropinesulfate, atropine oxide, methylatropine nitrate, homatropinehydrobromide, hyoscyamine (d, l) hydrobromide, scopolamine hydrobromide,ipratropium bromide, oxitropium bromide, tiotropium bromide,methantheline, propantheline bromide, anisotropine methyl bromide,clidinium bromide, copyrrolate (Robinul), isopropamide iodide,mepenzolate bromide, tridihexethyl chloride (Pathilone), hexocycliummethylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like, or a pharmaceutically acceptable saltthereof; or, for those compounds listed as a salt, alternatepharmaceutically acceptable salt thereof.

Representative antihistamines (i.e., H₁-receptor antagonists) that canbe used in combination with the compounds of this invention include, butare not limited to, ethanolamines, such as carbinoxamine maleate,clemastine fumarate, diphenylhydramine hydrochloride and dimenhydrinate;ethylenediamines, such as pyrilamine amleate, tripelennaminehydrochloride and tripelennamine citrate; alkylamines, such aschlorpheniramine and acrivastine; piperazines, such as hydroxyzinehydrochloride, hydroxyzine pamoate, cyclizine hydrochloride, cyclizinelactate, meclizine hydrochloride and cetirizine hydrochloride;piperidines, such as astemizole, levocabastine hydrochloride, loratadineor its descarboethoxy analogue, terfenadine and fexofenadinehydrochloride; azelastine hydrochloride; and the like, or apharmaceutically acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically acceptable salt thereof.

Suitable doses for the other therapeutic agents administered incombination with a compound of the invention are in the range of about0.05 g/day to about 100 mg/day.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

FORMULATION EXAMPLE A

A dry powder for administration by inhalation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 mg Lactose  25 mg

Representative Procedure: The compound of the invention is micronizedand then blended with lactose. This blended mixture is then loaded intoa gelatin inhalation cartridge. The contents of the cartridge areadministered using a powder inhaler.

FORMULATION EXAMPLE B

A dry powder formulation for use in a dry powder inhalation device isprepared as follows:

Representative Procedure: A pharmaceutical composition is preparedhaving a bulk formulation ratio of micronized compound of the inventionto lactose of 1:200. The composition is packed into a dry powderinhalation device capable of delivering between about 10 μg and about100 μg of the compound of the invention per dose.

FORMULATION EXAMPLE C

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

Representative Procedure: A suspension containing 5 wt. % of a compoundof the invention and 0.1 wt. % lecithin is prepared by dispersing 10 gof the compound of the invention as micronized particles with mean sizeless than 10 μm in a solution formed from 0.2 g of lecithin dissolved in200 mL of demineralized water. The suspension is spray dried and theresulting material is micronized to particles having a mean diameterless than 1.5 μm. The particles are loaded into cartridges withpressurized 1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE D

A pharmaceutical composition for use in a metered dose inhaler isprepared as follows:

Representative Procedure: A suspension containing 5% compound of theinvention, 0.5% lecithin, and 0.5% trehalose is prepared by dispersing 5g of active ingredient as micronized particles with mean size less than10 m in a colloidal solution formed from 0.5 g of trehalose and 0.5 g oflecithin dissolved in 100 mL of demineralized water. The suspension isspray dried and the resulting material is micronized to particles havinga mean diameter less than 1.5 μm. The particles are loaded intocanisters with pressurized 1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE E

A pharmaceutical composition for use in a nebulizer inhaler is preparedas follows:

Representative Procedure: An aqueous aerosol formulation for use in anebulizer is prepared by dissolving 0.1 mg of the compound of theinvention in 1 mL of a 0.9% sodium chloride solution acidified withcitric acid. The mixture is stirred and sonicated until the activeingredient is dissolved. The pH of the solution is adjusted to a valuein the range of from 3 to 8 by the slow addition of NaOH.

FORMULATION EXAMPLE F

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 250 mg Lactose(spray-dried) 200 mg Magnesium stearate  10 mg

Representative Procedure: The ingredients are thoroughly blended andthen loaded into a hard gelatin capsule (460 mg of composition percapsule).

FORMULATION EXAMPLE G

A suspension for oral administration is prepared as follows:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

Representative Procedure: The ingredients are mixed to form a suspensioncontaining 100 mg of active ingredient per 10 mL of suspension.

FORMULATION EXAMPLE H

An injectable formulation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 g Sodium acetate buffersolution (0.4M) 2.0 mL HCl (0.5N) or NaOH (0.5N) q.s. to pH 4 Water(distilled, sterile) q.s. to 20 mL

Representative Procedure: The above ingredients are blended and the pHis adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

Utility

The compounds of this invention possess both β₂ adrenergic receptoragonist and muscarinic receptor antagonist activity and therefore, suchcompounds are expected to be useful for treating medical conditionsmediated by β₂ adrenergic receptors or muscarinic receptors, i.e.,medical conditions that are ameliorated by treatment with a β₂adrenergic receptor agonist or a muscarinic receptor antagonist. Suchmedical conditions include, by way of example, pulmonary disorders ordiseases associated with reversible airway obstruction, such as chronicobstructive pulmonary disease (e.g., chronic and wheezy bronchitis andemphysema), asthma, pulmonary fibrosis and the like. Other conditionswhich may be treated include premature labor, depression, congestiveheart failure, skin diseases (e.g., inflammatory, allergic, psoriaticand proliferative skin diseases, conditions where lowering pepticacidity is desirable (e.g., peptic and gastric ulceration) and musclewasting disease.

Accordingly, in one embodiment, this invention is directed to a methodfor treating a pulmonary disorder, the method comprising administeringto a patient in need of treatment a therapeutically effective amount ofa compound of this invention or a pharmaceutically acceptable salt orsolvate or stereoisomer thereof. When used to treat a pulmonarydisorder, the compounds of this invention will typically be administeredby inhalation in multiple doses per day, in a single daily dose or asingle weekly dose. Generally, the dose for treating a pulmonarydisorder will range from about 10 μg/day to about 200 μg/day.

When administered by inhalation, the compounds of this inventiontypically have the effect of providing bronchodilation. Accordingly, inanother of its method aspects, this invention is directed to a method ofproducing bronchodilation in a patient, the method comprisingadministering to the patient requiring bronchodilation abronchodilation-producing amount of a compound of this invention or apharmaceutically acceptable salt or solvate or stereoisomer thereof.Generally, the dose for providing bronchodilation will range from about10 μg/day to about 200 μg/day.

In one embodiment, this invention is directed to a method of treatingchronic obstructive pulmonary disease or asthma, the method comprisingadministering to a patient in need of treatment a therapeuticallyeffective amount of a compound of this invention or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof. When used to treat aCOPD or asthma, the compounds of this invention will typically beadministered by inhalation in multiple doses per day or in a singledaily dose. Generally, the dose for treating COPD or asthma will rangefrom about 10 μg/day to about 200 μg/day.

As used herein, COPD includes chronic obstructive bronchitis andemphysema (see, for example, Barnes, Chronic Obstructive PulmonaryDisease, N Engl J Med 2000: 343:269-78).

When used to treat a pulmonary disorder, the compounds of this inventionare optionally administered in combination with other therapeuticagents. In particular, by combining the compounds of this invention witha steroidal anti-inflammatory agent (e.g. a corticosteroid), thepharmaceutical compositions of this invention can provide tripletherapy, i.e., β₂ adrenergic receptor agonist, muscarinic receptorantagonist and anti-inflammatory activity, using only two activecomponents. Since pharmaceutical compositions containing two activecomponents are typically easier to formulate compared to compositionscontaining three active components, such two component compositionsprovide a significant advantage over compositions containing threeactive components. Accordingly, in a particular embodiment, thepharmaceutical compositions and methods of this invention furthercomprise a therapeutically effective amount of a steroidalanti-inflammatory agent.

Compounds of this invention exhibit both muscarinic receptor antagonistand β₂ adrenergic receptor agonist activity. Accordingly, among otherproperties, compounds of particular interest are those that demonstratean inhibitory constant K_(i) value for binding at the M₃ muscarinicreceptor and an EC₅₀ value for β₂ adrenergic receptor agonist activityof less than about 100 nM; particularly less than 10 nM. Among thesecompounds, compounds of special interest include those having muscarinicactivity, expressed in terms of the inhibitory constant K_(i) forbinding at the M₃ muscarinic receptor, that is about equal to thecompound's β₂ adrenergic agonist activity, expressed in terms of thehalf maximal effective concentration EC₅₀, as determined in the in vitroassays described herein, or in similar assays. For example, compounds ofparticular interest are those having a ratio of the inhibitory constantK_(i) for the M₃ muscarinic receptor to the EC₅₀ for the β₂ adrenergicreceptor ranging from about 30:1 to about 1:30; including about 20:1 toabout 1:20; such as about 10:1 to about 1:10.

Since compounds of this invention possess both β₂ adrenergic agonistactivity and muscarinic receptor antagonist activity, such compounds arealso useful as research tools for investigating or studying biologicalsystems or samples having β₂ adrenergic receptors or muscarinicreceptors or combinations thereof, or for discovering new compoundshaving both β₂ adrenergic agonist activity and muscarinic receptorantagonist activity. Such biological systems or samples may comprise β₂adrenergic receptors and/or muscarinic receptors. Any suitablebiological system or sample having β₂ adrenergic and/or muscarinicreceptors may be employed in such studies which may be conducted eitherin vitro or in vivo. Representative biological systems or samplessuitable for such studies include, but are not limited to, cells,cellular extracts, plasma membranes, tissue samples, mammals (such asmice, rats, guinea pigs, rabbits, dogs, pigs, etc.), and the like.

In this embodiment, a biological system or sample comprising a β₂adrenergic receptor or a muscarinic receptor or a combination thereof iscontacted with a β₂ adrenergic receptor-agonizing or muscarinicreceptor-antagonizing amount of a compound of this invention. Theresponse of the biological system or sample to the compound is thenmeasured, or the effects of the compound on the biological system orsample are then determined using conventional procedures and equipment,such as radioligand binding assays and functional assays. Suchfunctional assays include ligand-mediated changes in intracellularcyclic adenosine monophosphate (cAMP), ligand-mediated changes inactivity of the enzyme adenylyl cyclase (which synthesizes cAMP),ligand-mediated changes in incorporation of guanosine5′-O-(-thio)triphosphate ([³⁵S]GTP S) into isolated membranes viareceptor catalyzed exchange of [³⁵S]GTP S for GDP, ligand-mediatedchanges in free intracellular calcium ions (measured, for example, witha fluorescence-linked imaging plate reader or FLIPR® from MolecularDevices, Inc.). A compound of this invention will agonize or causeactivation of a β₂ adrenergic receptor and antagonize or decrease theactivation of muscarinic receptors in any of the functional assayslisted above, or assays of a similar nature. The amount of compound usedin these studies will typically range from about 0.1 nanomolar to about100 nanomolar.

Additionally, the compounds of this invention can be used as researchtools for discovering new compounds that have both a β₂ adrenergicreceptor agonist and muscarinic receptor antagonist activity. In thisembodiment, a β₂ adrenergic receptor and muscarinic receptor bindingdata (for example, as determined by in vitro radioligand displacementassays) for a test compound or a group of test compounds is compared tothe β₂ adrenergic receptor and muscarinic receptor binding data for acompound of this invention to identify those test compounds that haveabout equal or superior β₂ adrenergic receptor and/or muscarinicreceptor binding, if any. This aspect of the invention includes, asseparate embodiments, both the generation of comparison data (using theappropriate assays) and the analysis of the test data to identify testcompounds of interest.

The properties and utility of the compounds of this invention can bedemonstrated using various in vitro and in vivo assays well-known tothose skilled in the art. For example, representative assays aredescribed in further detail in the following Examples.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of this invention. These specific embodiments,however, are not intended to limit the scope of this invention in anyway unless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard meaning:

AC adenylyl cyclase Ach acetylcholine ATCC American Type CultureCollection BSA bovine serum albumin cAMP 3′-5′ cyclic adenosinemonophosphate CHO Chinese hamster ovary cM₅ cloned chimpanzee M₅receptor DCM dichloromethane (i.e., methylene chloride) DIPEAN,N-diisopropylethylamine dPBS Dulbecco's phosphate buffered saline DMEMDulbecco's Modified Eagle's Medium DMSO dimethyl sulfoxide EDTAethylenediaminetetraacetic acid Emax maximal efficacy EtOAc ethylacetate EtOH ethanol FBS fetal bovine serum FLIPR fluorometric imagingplate reader Gly glycine HATUO-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate HBSS Hank's buffered salt solution HEK humanembryonic kidney cells HEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hM₁ cloned human M₁receptor hM₂ cloned human M₂ receptor hM₃ cloned human M₃ receptor hM₄cloned human M₄ receptor hM₅ cloned human M₅ receptor HPLChigh-performance liquid chromatography IBMX 3-isobutyl-1-methylxanthine% Eff % efficacy PBS phosphate buffered saline PyBOPbenzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate rpmrotations per minute TFA trifluoroacetic acid THF tetrahydrofuran Tristris(hydroxymethyl)aminomethane

Unless noted otherwise, reagents, starting materials and solvents werepurchased from commercial suppliers (such as Aldrich, Fluka, Sigma andthe like) and were used without further purification.

In the examples described below, HPLC analysis was conducted using anAgilent (Palo Alto, Calif.) Series 1100 instrument with Zorbax Bonus RP2.1×50 mm columns, supplied by Agilent, (a C14 column), having a 3.5micron particle size. Detection was by UV absorbance at 214 nm. HPLC10-70 data was obtained with a flow rate of 0.5 mL/minute of 10%-70% Bover 6 minutes. Mobile phase A was 2%-98%-0.1% ACN—H₂O-TFA; and mobilephase B was 90%-10%-0.1% ACN—H₂O-TFA. Using the mobile phases A and Bdescribed above, HPLC 5-35 data and HPLC 10-90 data were obtained with a5 minute gradient.

Liquid chromatography mass spectrometry (LCMS) data were obtained withan Applied Biosystems (Foster City, Calif.) model API-150EX instrument.LCMS 10-90 data was obtained with a 10%-90% mobile phase B over a 5minute gradient.

Small scale purification was conducted using an API 150EX PrepWorkstation system from Applied Biosystems. The mobile phase was A:water+0.05% v/v TFA; and B: acetonitrile+0.05% v/v TFA. For arrays(typically about 3 to 50 mg recovered sample size) the followingconditions were used: 20 mL/min flow rate; 15 min gradients and a 20mm×50 mm Prism RP column with 5 micron particles (ThermoHypersil-Keystone, Bellefonte, Pa.). For larger scale purifications(typically greater than 100 mg crude sample), the following conditionswere used: 60 mL/min flow rate; 30 min gradients and a 41.4 mm×250 mmMicrosorb BDS column with 10 micron particles (Varian, Palo Alto,Calif.).

The specific rotation for chiral compounds (indicated as [α]²⁰ _(D)) wasmeasured using a Jasco Polarimeter (Model P-1010) with a tungstenhalogen light source and a 589 nm filter at 20° C. Samples of testcompounds were typically measured at 1 mg/mL water.

Example 1 Biphenyl-2-ylcarbamic Acid1-{9-[2-(4-Hydroxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)-ethylamino]nonyl}piperidin-4-ylEster Bis(trifluoroacetate) Salt Step 1—Biphenyl-2-ylcarbamic AcidPiperidin-4-yl Ester

Biphenyl-2-isocyanate (97.5 g, 521 mmol) and4-hydroxy-1-benzylpiperidine (105 g, 549 mmol), bothcommercially-available from Aldrich, Milwaukee, Wis., were heatedtogether at 70° C. for 12 h, during which time the formation ofbiphenyl-2-ylcarbamic acid 1-benzylpiperidin-4-yl ester was monitored byLCMS. The reaction mixture was then cooled to 50° C. and ethanol (1 L)was added, and then 6M hydrochloric acid (191 mL) was added slowly. Thereaction mixture was then cooled to ambient temperature and ammoniumformate (98.5 g, 1.56 mol) was added and nitrogen gas was bubbledthrough the solution vigorously for 20 min. Palladium (10 wt. % (drybasis) on activated carbon) (20 g) was then added. The reaction mixturewas heated at 40° C. for 12 h and then filtered through a pad of Celite.The solvent was then removed under reduced pressure and 1M hydrochloricacid (40 mL) was added to the crude residue. Sodium hydroxide (10N) wasthen added to adjust the pH to 12. The aqueous layer was extracted withethyl acetate (2×150 mL) and dried (magnesium sulfate), and then thesolvent was removed under reduced pressure to give the title compound(155 g, 100% yield). HPLC (10-70) R_(t)=2.52; MS m/z: [M+H⁺] calc'd forC₁₈H₂₀N₂O₂ 297.15. found 297.3.

Step 2—Biphenyl-2-ylcarbamic Acid 1-(9-Hydroxynonyl)piperidin-4-yl Ester

A solution of biphenyl-2-ylcarbamic acid piperidin-4-yl ester (5 g, 16.9mmol), 9-bromo-1-nonanol (4.9 g, 22 mmol) and N,N-diisopropylethylamine(8.8 mL, 50.7 mmol) in acetonitrile (100 mL) was heated to 60° C. for 12hours. The reaction mixture was cooled and concentrated to dryness. Theresidue was dissolved in dichloromethane (50 mL) and this solution waswashed with 0.05N HCl (50 mL) and brine and then dried over magnesiumsulfate. The solvent was evaporated to yield the title compound, (6 g,81% yield) which was used without further purification in the next step.MS m/z: [M+H⁺] calcd for C₂₇H₃₈N₂O₃ 439.3. found 439.3.

Step 3—Biphenyl-2-ylcarbamic Acid 1-(9-Oxononyl)piperidin-4-yl Ester

A solution of the product of Step 2 (6 g, 13.7 mmol),N,N-diisopropylethylamine (7.15 mL, 41.1 mmol) and dimethyl sulfoxide(20 mL) was in dichloromethane (100 mL) was cooled to 0° C. After 15minutes, pyridine sulfur trioxide (6.54 g, 41.1 mmol) was added in twoportions to the cooled reaction mixture. After 2 hours at 0° C., thereaction was quenched with water (50 mL). The organic layer was thenwashed with water (3×50 mL) and concentrated to yield the title compound(5.8 g) which was used without further purification in the next step. MSm/z: [M+H⁺] calcd for C₂₇H₃₆N₂O₃ 437.3. found 437.4.

Step 4—7-(2-Aminoethyl)-4-methoxy-1,3-benzothiazol-2(3H)-oneHydrochloride (a) 7-Acetonitrile-2,4-dimethoxybenzothiazole

The title compound was prepared using the procedures described in J.Weinstock et al., J. Med. Chem., 1987, 30, 1166-1176. Following theprocedures in Weinstock at page 1173 and using2-methoxy-5-methylphenylthiourea (Lancaster Synthesis, Ltd., Windham,N.H.), the title compound was prepared with the following minormodifications in the synthesis of2,4-dimethoxybenzothiazole-7-acetonitrile: (1)2,2′-azobisisobutyronitrile (Aldrich, Milwaukee, Wis.) was substitutedfor benzoyl peroxide; and (2) the reaction mixture was refluxed undernitrogen for 30 minutes instead of being irradiated with a 150-Wtungsten lamp, and then the reaction mixture was cooled to 10° C. andfiltered.

(b) 7-(2-Aminoethyl)-4-methoxy-3H-benzothiazol-2-one

The title compound was prepared using the procedure described in J.Weinstock et al., J. Med. Chem., 1987, 30, 1166-1176 on page 1173. MSm/z: [M+H⁺] calcd for C₁₀H₁₂N₂O₂S 225.1. found 225.1. ¹H NMR (CDCl₃)δ=2.60 (t, 2 H, CH₂), 2.80 (t, 2 H, CH₂), 3.60 (s, 3 H, OCH₃), 6.82 (dd,2 H Ar).

Step 5—Biphenyl-2-ylcarbamic Acid1-{9-[2-(4-Methoxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)-ethylamino]nonyl}piperidin-4-ylEster

The product of Step 3 (91 mg, 0.35 mmol) was added to a mixture of theproduct of Step 4 (153 mg, 0.35 mmol) and N,N-diisopropylethylamine(0.061 mL, 0.35 mmol) in dichloromethane (1.75 mL), and the resultingmixture was stirred at room temperature for 2 hours. Sodiumtriacetoxyborohydride (88 mg, 0.42 mmol) was added and the mixture wasstirred at room temperature for 12 hours, at which time the reaction wasdetermined to be complete by LCMS (10-90) analysis. The reaction mixturewas then quenched with 6 N ammonium chloride solution (2 mL) and theorganic layer was separated. The organic layer was washed with brine,dried over magnesium sulfate and concentrated to give the title compound(145 mg). MS m/z: [M+H⁺] calcd for C₃₇H₄₈N₄O₄S 645.3. found 645.8.

Step 6—Biphenyl-2-ylcarbamic Acid1-{9-[2-(4-Hydroxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)-ethylamino]nonyl}piperidin-4-ylEster Bis(trifluoroacetate) Salt

A solution of the product of Step 5 (138 mg, 0.21 mmol) indichloromethane (1 mL) was cooled to −10° C. and 1.0 M boron tribromidein dichloromethane (1.1 mL) was added. After ten minutes, the ice bathwas removed and the reaction mixture was allowed to slowly warm to roomtemperature. After 3 hours, the reaction was determined to be completeby MS analysis. The reaction mixture was then quenched with methanol (1mL) and concentrated in vacuo. The residue was purified by HPLC (5-35)to give the title compound (9.8 mg, 98% pure). HPLC (10-70) R_(t)=2.9;MS m/z: [M+H⁺] calcd for C₃₆H₄₆N₄O₄S 631.3. found 631.8.

Example 2 Biphenyl-2-ylcarbamic Acid1-[2-(4-{[2-(4-Hydroxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)ethylamino]methyl}phenylcarbamoyl)ethyl]-piperidin-4-ylEster Bis(trifluoroacetate) Salt Step1—N-(4-Hydroxymethylphenyl)acrylamide

p-Aminobenzyl alcohol (12.31 g, 100 mmol) was dissolved in a mixture ofdichloromethane (200 mL) and tetrahydrofuran (20 mL) containingN,N-diisopropylethylamine (35 mL, 200 mmol). The resulting homogeneoussolution was then cooled to 0° C. and acryloyl chloride (8.2 mL, 100mmol) was added drop-wise over a 30-minute period while keeping theinternal temperature of the reaction mixture below 20° C. The reactionmixture was stirred at 0° C. for about 60 minutes. The reaction mixturewas poured into ice-cold 1M hydrochloric acid (0.6 L) and the organiclayer was separated and evaporated to dryness to give a crude material(5 g) consisting primarily of the bis-acylated side product. The acidicaqueous layer was then extracted with ethyl acetate (2×200 mL) and thecombined ethyl acetate layers were dried with sodium sulfate, filteredand concentrated under reduced pressure to provide a residue.Trituration of the residue with ethyl acetate yielded the title compound(10.5 g, 98.5% pure). An additional 2 g of the title compound wereobtained by trituration of the crude material obtained from thedichloromethane layer.

Step 2—Biphenyl-2-ylcarbamic Acid1-[2-(4-Hydroxymethylphenylcarbamoyl)ethyl]-piperidin-4-yl Ester

The product of Step 1 (10 g, 57 mmol) and biphenyl-2-ylcarbamic acidpiperidin-4-yl ester from Example 1, Step 1 (17.8 g, 60 mmol) weredissolved in a mixture of methanol (100 mL) and dichloromethane (100 mL)and the resulting mixture was heated at 55° C. (reflux) for 18 hours.Most of the solvent was then removed under reduced pressure and theresulting residue was triturated with ethyl acetate (200 mL) to give asolid that was isolated by filtration. The solid was dried under vacuumto give the title compound (25 g).

Step 3—Biphenyl-2-ylcarbamic Acid1-[2-(4-Formylphenylcarbamoyl)ethyl]piperidin-4-yl Ester

The product of Step 2 (20 g, 42.3 mmol) was dissolved in anhydrousdichloromethane (200 mL) containing DMSO (18 mL, 254 mmol) andN,N-diisopropylethylamine (37 mL, 211.5 mmol). The resulting homogeneoussolution was cooled to −20° C. and then sulfur trioxide pyridine complex(20.2 g) was added in portions over a 30-minute period while maintainingthe internal temperature of the reaction mixture below −10° C. Thereaction mixture was then stirred at −10° C. for about 30 minutes. Thereaction mixture was then poured into an ice-cold mixture of 1Mhydrochloric acid (100 mL) and water (500 mL) (the mixture had a pH ofabout 6). The mixture was extracted with dichloromethane (300 mL) andthe dichloromethane layer was then washed with brine (200 mL), driedwith sodium sulfate and concentrated under reduced pressure. Someprecipitation was observed during solvent removal. Ethyl acetate (100mL) was added to the resulting thick slurry. The resulting solid wasisolated by filtration and dried under vacuum to give the title compound(11 g, 99% purity by HPLC). The slurry solvent was concentrated underreduced pressure to give additional solid that was isolated byfiltration and dried under vacuum to give the title compound (5.4 g, 96%purity by HPLC). HPLC (2%-70% acetonitrile in water/0.1% TFA);R_(t)=5.7. MS m/z: [M+H⁺] calcd for C₂₈H₂₉N₃O₄ 472.2. found 472.0.

Step 4—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[2-(4-Methoxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)ethylamino]methyl}phenylcarbamoyl)ethyl]-piperidin-4-ylEster

The product of Step 3 (163 mg, 0.35 mmol) was added to a mixture of theproduct of Example 1, Step 4 (100 mg, 0.38 mmol) andN,N-diisopropylethylamine (0.067 mL, 0.38 mmol) in dichloromethane (1.75mL) and the resulting mixture was stirred at room temperature for 2hours. Sodium triacetoxyborohydride (122 mg, 0.58 mmol) was added andthe resulting mixture was stirred at room temperature for about 2 hours.The reaction mixture was quenched with 6 N ammonium chloride (2 mL) andthe organic layer was separated. The organic layer was washed withbrine, dried over magnesium sulfate and concentrated under vacuum togive the title compound as a solid (127 mg). MS m/z: [M+H⁺] calcd forC₃₈H₄₁N₅O₅S 680.3. found 680.8.

Step 5—Biphenyl-2-ylcarbamic Acid1-[2-(4-{[2-(4-Hydroxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)ethylamino]methyl}phenylcarbamoyl)ethyl]-piperidin-4-ylEster Bis(trifluoroacetate) Salt

A solution of the product of Step 4 (127 mg, 0.19 mmol) indichloromethane (1 mL) was cooled to −10° C. and a 1 M solution of borontribromide in dichloromethane (0.94 mL) was added. After 10 minutes, theice bath was removed and the reaction mixture was allowed to slowly warmto room temperature. After 3 hours, the reaction was determined to becomplete by MS analysis. The reaction mixture was quenched by slowlyadding methanol (1 mL) and then concentrated in vacuo. The crude mixturewas then purified using HPLC (5-35) to give the title compound as apowder (11.7 mg, 98% purity).

MS m/z: [M+H⁺] calcd for C₃₇H₃₉N₅O₅S 666.3. found 666.5.

Example 33-[4-(3-Biphenyl-2-ylureido)piperidin-1-yl]-N-(4-{[2-(4-hydroxy-2-oxo-2,3-dihydro-benzothiazol-7-yl)ethylamino]methyl}-phenyl)propionamideStep 1—N-1,1′-Biphenyl-2-yl-N′-4-piperidinylurea (a)N-1,1′-Biphenyl-2-yl-N′-4-(1-benzyl)piperidinylurea

Biphenyl-2-isocyanate (50 g, 256 mmol) was dissolved in acetonitrile(400 mL) at ambient temperature. After cooling to 0° C., a solution of4-amino-N-benzylpiperidine (48.8 g, 256 mmol) in acetonitrile (400 mL)was added over 5 minutes. A precipitate was observed immediately. After15 minutes, acetonitrile (600 mL) was added, and the resultant viscousmixture was stirred for 12 hours at 35° C. The solids were filtered andwashed with cold acetonitrile and then dried under vacuum to give thetitle compound (100 g, 98% yield). MS m/z: [M+H⁺] calcd for C₂₅H₂₇N₃O386.22. found 386.3.

(b) N-1,1′-Biphenyl-2-yl-N′-4-piperidinylurea

The product of Step (a) (20 g, 52 mmol) was dissolved in a mixture ofanhydrous methanol and anhydrous DMF (3:1 v/v, 800 mL). Aqueoushydrochloric acid (0.75 mL of 37% conc. solution, 7.6 mmol) was addedand nitrogen gas was bubbled through the solution vigorously for 20minutes. Pearlman's catalyst (Pd(OH)₂, 5 g) was added under a stream ofnitrogen, before placing the reaction mixture under a hydrogenatmosphere (balloon). The reaction mixture was allowed to stir for 4days and was then passed twice through pads of Celite to remove thecatalyst. The solvent was then removed under reduced pressure to givethe title compound (13 g, 85% yield). MS m/z: [M+H⁺] calcd for C₁₈H₂₁N₃O296.2. found 296.0.

Alternatively, N-1,1′-biphenyl-2-yl-N′-4-piperidinylurea was synthesizedby heating together biphenyl-2-isocyanate (50 g, 256 mmol) and4-amino-1-benzylpiperidine (51.1 g, 269 mmol) at 70° C. for 12 hours(monitored by LCMS analysis). The reaction mixture was cooled to 50° C.and ethanol (500 mL) added, followed by slow addition of 6M hydrochloricacid (95 mL). The reaction mixture was cooled to room temperature.Ammonium formate (48.4 g, 768 mmol) was added to the reaction mixtureand nitrogen gas bubbled through the solution vigorously for 20 minutes,before adding palladium (10 wt. % (dry basis) on activated carbon) (10g). The reaction mixture was heated at 40° C. for 12 hours, beforefiltering through a pad of Celite and the solvent removed under reducedpressure. To the crude residue was added 1M hydrochloric acid (20 mL)and 10N sodium hydroxide was added to adjust the pH to 12. The aqueouslayer was extracted with ethyl acetate (2×80 mL), dried (magnesiumsulfate) and the solvent removed under reduced pressure to give thetitle compound as a solid (71.7 g, 95% yield). MS m/z: [M+H⁺] calcd forC₁₈H₂₁N₃O 296.2. found 296.0.

Step 2—N-(4-(1,3-Dioxolan-2-yl)phenyl)acrylamide (a)2-(4-Nitrophenyl)-[1,3]-dioxolane

In a 3-neck round-bottom flask equipped with Dean-Stark apparatus,reflux condenser and mechanical stirrer, p-nitrobenzaldehyde (101.5 g,672 mmol), ethylene glycol (112 mL, 2.0 mol), and p-toluene sulfonicacid (12.8 g, 67.2 mmol, 10% mol) were suspended in toluene (800 mL) andthen heated at 120° C. for 4 hours. The reaction mixture was cooled toroom temperature and the toluene was removed under reduced pressure.Saturated aqueous sodium bicarbonate solution (800 mL) was added, andthe resulting slurry was stirred at room temperature for 15 minutes, andthen filtered and dried under vacuum to give the title compound (121.8g) as a solid. ¹H NMR (DMSO-d₆): δ=8.12 (d, 2H), 7.59 (d 2H), 5.78 (s,1H), 3.8-4.0 (m, 4H).

(b) 4-([1,3]-Dioxolan-2-yl)phenylamine

The product of Step (a) (10 g, 51 mmol) was dissolved in a mixture oftetrahydrofuran (50 mL) and ethanol (50 mL) and then hydrogenated at 50psi for 18 hours using platinum oxide catalyst (PtO₂) (116 mg, 0.51mmol). The reaction mixture was filtered through Celite and then thesolvent was removed under reduced pressure to give the title compound (8g), which was used in the next step without further purification. ¹H NMR(DMSO-d₆): δ=6.98 (d, 2H), 6.42 (d, 2H), 5.39 (s, 1H), 5.08 (s, 2H)3.7-3.9 (m, 4H).

(c) N-(4-([1,3]Dioxolan-2-yl)phenyl)acrylamide

The product from Step (b) (8 g, 48.5 mmol)) and triethylamine (10.1 mL,72.75 mmol) were dissolved in dichloromethane (100 mL). The resultinghomogeneous solution was cooled to 0° C. and acryloyl chloride (4.81 mL,58.2 mmol was added dropwise. The reaction mixture was stirred at 0° C.for 1 hour and then quenched with water (100 mL). The organic layer waswashed with water (50 mL), dried with sodium sulfate and concentratedunder reduced pressure to remove most of the dichloromethane. Ethylacetate (100 mL) was added to the residue and the resulting precipitatewas collected by filtration and then dried under vacuum to afford thetitle compound (8.5 g). ¹H NMR (DMSO-d₆): δ=10.10 (s, 1H), 7.61 (d, 2H),7.25 (d, 2H), 6.1-6.4 (m, 2H), 5.62 (d, 1H), 5.58 (s, 1H), 3.7-4.0 (m,4H).

Step3—3-[4-(3-Biphenyl-2-ylureido)piperidin-1-yl]-N-(4-([1,3]dioxolan-2-yl)phenyl)-propionamide

The product of Step 1 (543 mg, 1 mmol) was added to a solution of theproduct of Step 2 (385 mg, 1.7 mmol) in a mixture of methanol (3 mL) anddichloromethane (3 mL) and the resulting mixture heated to reflux for 12hours. The reaction mixture was then cooled and concentrated undervacuum. The residue was triturated with ethyl acetate and the resultingprecipitate was isolated by filtration to give the title compound (731mg) as a solid. MS m/z: [M+H⁺] calcd for C₃₀H₃₄N₄O₄ 515.3. found 515.5.

Step4—3-[4-(3-Biphenyl-2-ylureido)piperidin-1-yl]-N-(4-formylphenyl)propionamide

To a solution of the product of Step 3 (731 mg, 1.4 mmol) in methanolwas added 1 M hydrochloric acid (2 mL) and the resulting mixture wasstirred at room temperature for two hours. The crude reaction mixturewas then concentrated under vacuum and then diluted withdichloromethane. The organic layer was washed with saturated sodiumbicarbonate (2×5 mL) and then brine (5 mL). The organic layer was driedover magnesium sulfate, filtered, and concentrated to give the titlecompound (600 mg) as an oil. MS m/z: [M+H⁺] calcd for C₂₈H₃₀N₄O₃ 471.2.found 471.5.

Step5—3-[4-(3-Biphenyl-2-ylureido)piperidin-1-yl]-N-(4-{[2-(4-methoxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)ethylamino]methyl}phenyl)propionamide

The product of Example 1, Step 4 (131 mg, 0.58 mmol) was added to asolution of the product of Step 4 (183 mg, 0.39 mmol) andN,N-diisopropylethylamine (0.102 mL, 0.58 mmol) in a mixture ofdichloromethane (1 mL) and methanol (1 mL) and the resulting mixture wasstirred for 30 minutes at room temperature. After 30 minutes, sodiumtriacetoxyborohydride (123 mg, 0.58 mmol) was added and stirring wascontinued at room temperature. After 2 hours, the reaction mixture wasquenched with 6 N aqueous ammonium chloride (2 mL) and the organic layerwas separated. The organic layer was washed with saturated sodiumbicarbonate (2×5 mL) and then brine (5 mL). The organic layer was driedover magnesium sulfate, filtered, and then concentrated under vacuum togive the title compound (169 mg) as a solid. MS m/z: [M+H⁺] calcd forC₃₈H₄₂N₆O₄S 679.3. found 679.5.

Step6—3-[4-(3-Biphenyl-2-ylureido)piperidin-1-yl]-N-(4-{[2-(4-hydroxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)ethylamino]methyl}phenyl)propionamide

A solution of the product of Step 5 (169 mg, 0.25 mmol) indichloromethane (1.2 mL) was cooled in an ice/acetone bath. After about10 minutes, a 1.0 M solution of boron tribromide in dichloromethane (1.2mL) was slowly added to the reaction mixture while the reaction mixturewas stirred in the ice/acetone bath. After 30 minutes, the reactionmixture was removed from the ice bath and slowly warmed to roomtemperature. After 15 hours, the reaction mixture was slowly quenchedwith methanol (2 mL) and concentrated under vacuum. The reaction waspurified on a small-scale HPLC to give the title compound (8.7 mg, 71%purity). MS m/z: [M+H⁺] calcd for C₃₇H₄₀N₆O₄S 665.3. found 665.5. HPLC(10-70) R_(t)=2.97.

Example 4 Biphenyl-2-ylcarbamic Acid1-[2-(4-{2-[2-(4-Hydroxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)-ethylamino]ethyl}phenylcarbamoyl)ethyl]-piperidin-4-ylEster Bis(trifluoroacetate) Salt Step1—3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic Acid (a)Methyl 3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionate

Methyl 3-bromopropionate (553 μL, 5.07 mmol) was added to a stirredsolution of the product from Example 1, Step 1 (1.00 g, 3.38 mmol) anddiisopropylethylamine (1.76 mL, 10.1 mmol) in acetonitrile (34 mL) at50° C. and the reaction mixture was heated at 50° C. overnight. Thesolvent was then removed under reduced pressure, and the residue wasdissolved in dichloromethane (30 mL). The resulting solution was washedwith saturated aqueous sodium bicarbonate solution (10 mL), dried overmagnesium sulfate, filtered and concentrated under reduced pressure. Thecrude residue was purified by column chromatography to give the titlecompound (905 mg, 70% yield).

(b) 3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic Acid

A stirred solution of the product of Step (a) (902 mg, 2.37 mmol) andlithium hydroxide (171 mg, 7.11 mmol) in a 50% mixture oftetrahydrofuran (12 mL) and water (12 mL) was heated at 30° C. overnightand then acidified with concentrated hydrochloric acid. The resultingmixture was lyophilized to give the title compound (˜100% yield,containing some lithium chloride).

Step 2—Biphenyl-2-ylcarbamic Acid1-{2-[4-(2-Hydroxyethyl)phenylcarbamoyl]ethyl}-piperidin-4-yl Ester

4-Aminophenethyl alcohol (0.092 mg, 0.67 mmol) (Sigma Aldrich) was addedto a solution of the product of Step 1 (226 mg, 0.61 mmol) andN,N-diisopropylethylamine (0.161 mL, 0.67 mmol) in N,N-dimethylformamide(3.3 mL). The resulting mixture was stirred at room temperature for 45minutes. HATU (257 mg, 0.67 mmol) was then added and the reactionmixture was stirred at room temperature for 12 hours. The reactionmixture was then concentrated under vacuum to half its volume and thendiluted with dichloromethane. The organic layer was washed withsaturated sodium bicarbonate (2×5 mL) and then with brine (5 mL). Theorganic layer was dried over magnesium sulfate, filtered, andconcentrated to give the title compound (299 mg). MS m/z: [M+H⁺] calc'dfor C₂₉H₃₃N₃O₄ 488.3. found 488.3.

Step 3—Biphenyl-2-ylcarbamic Acid1-{2-[4-(2-Oxoethyl)phenylcarbamoyl]ethyl}-piperidin-4-yl Ester

A solution of the product of Step 2 (295 mg, 0.61 mmol) indichloromethane (3 mL) was cooled to −5° C. in an ice/water bath.Dimethyl sulfoxide (0.258 mL, 0.36 mmol) and N,N-diisopropylethylamine(0.316 mL, 1.8 mmol) were added and the reaction mixture was stirred for10 minutes at −5° C. Pyridine sulfur trioxide complex (289 mg, 1.8 mmol)was then added with stirring while maintaining the temperature of thereaction mixture at −5° C. After 2 hours, the reaction was complete asdetermined by MS analysis. The reaction mixture was then quenched withwater (5 mL) and the organic layer was washed with water (3×5 mL), driedover magnesium sulfate, filtered, and concentrated to give the titlecompound (176 mg) as a solid. MS m/z: [M+H⁺] calc'd for C₂₉H₃₁N₃O₄486.2. found 486.3.

Alternatively, biphenyl-2-ylcarbamic acid1-{2-[4-(2-oxoethyl)phenylcarbamoyl]-ethyl}piperidin-4-yl ester can besynthesized by substituting 4-amino phenethyl alcohol for p-aminobenzylalcohol as the starting material in Example 2, Step 1 and following thesynthetic procedures outlined in Example 2, Steps 1 to 3.

Step 4—Biphenyl-2-ylcarbamic Acid1-[2-(4-{2-[2-(4-Methoxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)-ethylamino]ethyl}phenylcarbamoyl)ethyl]piperidin-4-ylEster

The product of Step 3 (176 mg, 0.36 mmol) was added to a solution of theproduct of Example 1, Step 4 (106 mg, 0.47 mmol) in dichloromethane (2mL) and the resulting mixture was stirred at room temperature for 1hour. Sodium triacetoxyborohydride (84 mg, 0.40 mmol) was added and theresulting mixture was stirred at room temperature for 2 hours. Thereaction mixture was quenched with 6 N ammonium chloride (5 mL). Theorganic layer was separated and washed with saturated sodium bicarbonate(2×5 mL) and then with brine (5 mL). The organic layer was dried overmagnesium sulfate, filtered, and concentrated to give the title compoundas a solid (207 mg). MS m/z: [M+H⁺] calc'd for C₃₉H₄₃N₅O₅S 694.3. found694.3.

Step 5—Biphenyl-2-ylcarbamic Acid1-[2-(4-{2-[2-(4-Hydroxy-2-oxo-2,3-dihydrobenzothiazol-7-yl)-ethylamino]ethyl}phenylcarbamoyl)ethyl]piperidin-4-ylEster Bis(trifluoroacetate) Salt

A solution of the product of Step 4 (207 mg, 0.30 mmol) indichloromethane (1.5 mL) was cooled to 5° C. in an ice/water bath. Afterabout 10 minutes, a 1.0 M solution of boron tribromide indichloromethane (0.90 mL, 0.90 mmol) was added to the reaction mixture.After about 5.5 hours, the reaction mixture was removed from the icebath and stirred at room temperature. After 12 hours, the reactionmixture was concentrated to dryness and purified by HPLC to give thetitle compound (8 mg, 99% purity) as a bis(trifluoroacetic) acid salt.MS m/z: [M+H⁺] calc'd for C₃₈H₄₁N₅O₅S 680.3. found 680.0.

Additionally, other compounds of this invention can be prepared usingthe following intermediates.

Preparation 1

N-1,1′-Biphenyl-2-yl-N′-4-[1-(9-hydroxynonyl)]piperidinylurea

9-Bromo-1-nonanol (4.84 g, 21.7 mmol) was added to a stirred solution ofthe product of Example 3, Step 1 (5.8 g, 19.7 mmol) anddiisopropylethylamine (10.29 mL, 59.1 mmol) in acetonitrile (99 mL) at50° C. The reaction mixture was heated at 50° C. for 8 h. The reactionmixture was then allowed to cool and the solvent was removed underreduced pressure. The residue was dissolved in dichloromethane (100 mL),washed with saturated aqueous sodium bicarbonate (2×50 mL) and dried(magnesium sulfate). The solvent was removed under reduced pressure. Thecrude product was purified by flash chromatography(dichloromethane:methanol:ammonia system) to yield the title compound(7.1 g, 16.2 mmol, 82% yield).

Preparation 2

N-1,1′-Biphenyl-2-yl-N′-4-[1-(9-oxononyl)]piperidinylurea

Dimethyl sulfoxide (490 μL, 6.9 mmol), followed by diisopropylethylamine(324 μL, 3.45 mmol) was added to a solution of the product ofPreparation 1 (500 mg, 1.15 mmol) in dichloromethane (11.5 mL) at −10°C. under an atmosphere of nitrogen. The reaction mixture was stirred at−15° C. for 15 min, and then sulfur trioxide pyridine complex was addedportionwise (549 mg, 3.45 mmol). The reaction mixture was stirred at−15° C. for 1 h, and then water (10 mL) was added. The organic phase wasthen separated, washed with water (10 mL), and dried (sodium sulfate).The solvent was removed under reduced pressure to give the titlecompound (475 mg, 1.09 mmol, 95% yield). HPLC (10-70) R_(t)=3.39.

Preparation 3

N,N-(Di-tert-butoxycarbonyl)-9-bromononylamine

A solution of di-tert-butoxycarbonylamine (3.15 g, 14.5 mmol) inN,N-dimethylformamide (0.28 mL) was cooled to 0° C. for about 10 min.Sodium hydride, 60% in mineral oil (0.58 g, 14.5 mmol) was added and thereaction mixture was stirred at 0° C. for 10 min. The reaction mixturewas removed from the ice bath and allowed to warm to room temperaturefor about 30 min. The reaction mixture was then cooled back down to 0°C. and a solution of 1,9-dibromononane (2.46 mL, 12.1 mmol) indimethylformamide (100 mL) was added. The reaction mixture was stirredovernight at room temperature. After 24 h, MS analysis showed that thereaction was completed. The reaction mixture was concentrated to drynessand diluted with ethyl acetate (100 mL). The organic layer was washedwith saturated sodium bicarbonate (2×100 mL), brine (100 mL), dried(magnesium sulfate) and concentrated under reduced pressure to yield thecrude product, which was purified by chromatography on silica gel using5% ethyl acetate in hexanes to afford the title compound. MS m/z: [M+H⁺]calcd for C₁₉H₃₆N₁O₄Br 423.18. found 423.

Preparation 4

Biphenyl-2-ylcarbamic Acid1-(9-Di-tert-butoxycarbonylamino)nonyl]piperidin-4-yl Ester

A mixture of 1:1 acetonitrile and N,N-dimethylformamide (50 mL) wasadded to the products of Example 1, Step 1 (3.0 g, 10.1 mmol) andPreparation 3 (5.1 g, 12.2 mmol) and triethylamine (1.42 mL, 10.1 mmol).The reaction mixture was stirred at ambient temperature for 24 h and wasmonitored by LCMS analysis. The reaction mixture was then concentratedand diluted with ethyl acetate (50 mL). The organic layer was washedwith saturated sodium bicarbonate (2×50 mL) and brine (50 mL). Theorganic phase was then dried over magnesium sulfate and concentrated toyield 6.5 g of crude oil. The oil was purified by chromatography onsilica gel using 1:1 hexanes/ethyl acetate to provide the title compound(3 g). MS m/z: [M+H⁺] calcd for C₃₇H₅₅N₃O₆ 638.41. found 639.

Preparation 5

Biphenyl-2-ylcarbamic Acid 1-(9-Aminononyl)piperidin-4-yl Ester

Trifluoroacetic acid (11 mL) was added to a solution of the product ofPreparation 4 (7.2 g, 11.3 mmol) in dichloromethane (56 mL). After 2 h,LCMS analysis showed that the reaction was completed. The reactionmixture was then concentrated to dryness and diluted with ethyl acetate(75 mL). Sodium hydroxide (1N) was then added until the pH of themixture reached 14. The organic phase was then collected and washed withsaturated sodium bicarbonate (2×50 mL) and brine (50 mL). The organicphase was then dried over magnesium sulfate and concentrated to providethe title compound (5.5 g). MS m/z: [M+H⁺] calcd for C₂₇H₃₉N₃O₂ 438.30.found 439.

Preparation 6

Biphenyl-2-ylcarbamic Acid 1-(9-Oxononyl)piperidin-4-yl Ester (a)9-Bromononanal

To a 100-mL round-bottomed flask equipped with a magnetic stirrer,addition funnel and temperature controller, under nitrogen, was added9-bromononanal (8.92 g, 40 mmol) and dichloromethane (30 mL). Theresulting mixture was cooled to 5° C. and a solution of sodiumbicarbonate (0.47 g, 5.6 mmol) and potassium bromide (0.48 g, 4 mmol) inwater (10 mL) was added. 2,2,6,6-Tetramethyl-1-piperidinyloxy freeradical (TEMPO) (63 mg, 0.4 mmol) was added and then a 10 to 13% bleachsolution (27 mL) was added dropwise through the addition funnel at arate such that the temperature was maintained at about 8° C. (+/−2° C.)with an ice cold bath (over about 40 min.). After addition of the bleachwas complete, the mixture was stirred for 30 min. while maintaining thetemperature at about 0° C. A solution of sodium bisulfite (1.54 g) inwater (10 mL) was added and the resulting mixture was stirred at roomtemperature for 30 min. The layers of the mixture were then separated,and the milky aqueous layer was extracted with dichloromethane (1×20mL). The combined dichloromethane layers were then washed with water(1×30 mL), dried (MgSO₄), filtered and concentrated under reducedpressure to afford the title intermediate (8.3 g, 94% yield), which wasused without further purification in the next step.

(b) 9-Bromo-1,1-dimethoxynonane

To a 100 mL round-bottomed flask was added 9-bromononanal (7.2 g, 32.5mmol), methanol (30 mL) and trimethylorthoformate (4 mL, 36.5 mmol). Asolution of 4 N hydrochloric acid in dioxane (0.2 mL, 0.8 mmol) wasadded and the resulting mixture was refluxed for 3 h. The reactionmixture was then cooled to room temperature and solid sodium bicarbonate(100 mg, 1.2 mmol) was added. The resulting mixture was concentrated toone-fourth its original volume under reduced pressure and then ethylacetate (50 mL) was added. The organic layer was washed with water (2×40mL), dried (MgSO₄), filtered and concentrated under reduced pressure toafford the title intermediate (8.44 g, (97% yield)) as a liquid, whichas used in the next step without further purification.

(c) Biphenyl-2-ylcarbamic Acid 1-(9,9-Dimethoxynonyl)piperidin-4-ylEster

To a 50 mL three-necked, round-bottomed flask was addedbiphenyl-2-ylcarbamic acid piperidin-4-yl ester (1 g, 3.38 mmol) andacetonitrile (10 mL) to form a slurry. To this slurry was added9-bromo-1,1-dimethoxynonane (1.1 g, 1.3 mmol) and triethylamine (0.57 g,4.1 mmol) and the resulting mixture was heated at 65° C. for 6 h (thereaction was monitored by HPLC until starting material is <5%). Thereaction mixture was then cooled to room temperature at which time themixture formed a thick slurry. Water (5 mL) was added and the mixturewas filtered to collect the solid on a coarse fritted glass filer. Thesolid was washed with pre-mixed solution of acetonitrile (10 mL) andwater (5 mL) and then with another pre-mixed solution of acetonitrile(10 mL) and water (2 mL). The resulting solid was air dried to affordthe title intermediate (1.37 g, 84%, purity >96% by LC, ¹H NMR) as awhite solid.

(d) Biphenyl-2-ylcarbamic Acid 1-(9-Oxononyl)piperidin-4-yl Ester

To a 500 mL round-bottomed flask with a magnetic stirrer was addedbiphenyl-2-ylcarbamic acid 1-(9,9-dimethoxynonyl)piperidin-4-yl ester(7.7 g, 15.9 mmol) and then acetonitrile (70 mL) and aqueous 1Mhydrochloric acid (70 mL). The resulting mixture was stirred at roomtemperature for 1 h and then dichloromethane (200 mL) was added. Thismixture was stirred for 15 min. and then the layers were separated. Theorganic layer was dried (MgSO₄), filtered and concentrated under reducedpressure to afford the title intermediate (6.8 g).

Preparation 7

2-(N-Benzyloxycarbonyl-N-methylamino)ethanal (a)2-(N-Benzyloxycarbonyl-N-methylamino)ethanol

Benzyl chloroformate (19 g, 111.1 mmol) in THF (20 mL) was addeddropwise over 15 min to a stirred solution of 2-(methylamino)ethanol (10g, 133.3 mmol) in THF (100 mL) and aqueous sodium carbonate (100 mL) at0° C. The reaction mixture was stirred at 0° C. for 12 h and thenextracted with EtOAc (2×200 mL). The organic layer was washed withaqueous sodium carbonate (200 mL) and dried (potassium carbonate) andsolvent was removed under reduced pressure to give the title compound(22.5 g, 97% yield).

(b) 2-(N-Benzyloxycarbonyl-N-methylamino)ethanal

DMSO (71 mL, 1 mol) and DIPEA (87.1 mL, 0.5 mol) were added to a stirredsolution of the product of step (a) (20.9 g, 0.1 mol) in dichloromethane(200 mL) at −10° C. The reaction mixture was stirred at −10° C. for 15min and then sulfur trioxide pyridine complex (79.6 g, 0.5 mol) wasadded and the resulting mixture was stirred for 1 hour. The reactionmixture was quenched with addition of 1M hydrochloric acid (200 mL). Theorganic layer was separated and washed with saturated aqueous sodiumbicarbonate (100 mL), brine (100 mL), dried (potassium carbonate) andsolvent removed under reduced pressure to give the title compound (20.7g, ˜100% yield).

Preparation 8

Biphenyl-2-ylcarbamic Acid 1-[2-(Methylamino)ethyl]piperidin-4-yl Ester

To a stirred solution of the product of Preparation 7 (20.7 g, 100 mmol)and the product of Example 1, Step 1 (25 g, 84.7 mmol) in MeOH (200 mL)was added sodium triacetoxyborohydride (21.2 g, 100 mmol). The reactionmixture was stirred for 12 h at ambient temperature and then it wasquenched with 2M hydrochloric acid and the solvent was removed underreduced pressure. The residue was dissolved in ethyl acetate (200 mL)and washed with saturated aqueous sodium bicarbonate solution (100 mL)and brine (50 mL), and then dried (magnesium sulfate) and the solventwas removed under reduced pressure. The crude residue was purified bycolumn chromatography (50-90% EtOAc/hexanes) to givebiphenyl-2-ylcarbamic acid1-[2-(benzyloxycarbonyl-methylamino)ethyl]piperidin-4-yl ester as anoil.

The oil was dissolved in methanol (100 mL) and palladium (10 wt. % (drybasis) on activated carbon) (5 g) was added. The reaction mixture wasstirred under hydrogen (30 psi) for 12 h and then filtered throughCelite, which was washed with methanol, and solvent was evaporated togive the title compound (13.2 g, 44% yield).

Preparation 9

Biphenyl-2-ylcarbamic Acid1-{2-[(6-Bromohexanoyl)methylamino]ethyl}piperidin-4-yl Ester

6-Bromohexanoyl chloride (3.23 mL, 21.1 mmol) was added to a stirredsolution of the product of Preparation 8 (6.2 g, 17.6 mmol) and DIPEA(6.13 mL, 35.2 mmol) in dichloroethane (170 mL). The reaction mixturewas stirred for 1 hour and it was then diluted with EtOAc (250 mL) andwashed with saturated aqueous sodium bicarbonate solution (2×200 mL) andbrine (200 mL), and then dried (magnesium sulfate). The solvent wasremoved under reduced pressure to give the title compound (6.6 g, 73%yield).

Preparation 10

Biphenyl-2-ylcarbamic Acid1-[2-(4-(Aminomethyl)phenylcarbamoyl)-ethyl]piperidin-4-yl Ester

To a stirred solution of 4-(N-tert-butoxycarbonylaminomethyl)aniline(756 mg, 3.4 mmol), the product of Example 4, Step 1 (1.5 g, 4.08 mmol)and HATU (1.55 g, 4.08 mmol) in DMF (6.8 mL) was added DIPEA (770 μL,4.42 mmol). The reaction mixture was stirred at 50° C. overnight andthen the solvent was removed under reduced pressure. The resultingresidue was dissolved in dichloromethane (20 mL) and washed withsaturated aqueous sodium bicarbonate solution (10 mL). The organic phasewas then dried (magnesium sulfate) and the solvent was removed underreduced pressure. The crude product was purified by flash chromatography(5-10% MeOH/DCM) to give a solid, which was dissolved in TFA/DCM (25%,30 mL) and stirred at room temperature for 2 h. The solvent was thenremoved under reduced pressure and the crude residue was dissolved indichloromethane (30 mL) and washed with 1N sodium hydroxide (15 mL). Theorganic phase was separated, dried (magnesium sulfate) and the solventwas removed under reduced pressure to give the title compound (1.5 g,94% over 2 steps).

Preparation 11

Biphenyl-2-ylcarbamic Acid1-(2-tert-Butoxycarbonylaminoethyl)piperidin-4-yl Ester

To a stirred solution of the product of Example 1, Step 18 (2.00 g, 6.76mmol) and DIPEA (3.54 mL, 20.3 mmol) in acetonitrile (67.6 mL) at 50° C.was added 2-tert-butoxycarbonylaminoethyl bromide (1.82 g, 8.11 mmol)and the reaction mixture was heated at 50° C. overnight. The solvent wasthen removed under reduced pressure and the residue was dissolved indichloromethane (60 mL) and washed with saturated aqueous sodiumbicarbonate solution (30 mL). The organic phase was dried (magnesiumsulfate) and the solvent was removed under reduced pressure. The cruderesidue was purified by column chromatography (5% MeOH/DCM) to yield thetitle compound as a solid (2.32 g, 78% yield).

Preparation 12

Biphenyl-2-ylcarbamic Acid 1-(2-Aminoethyl)piperidin-4-yl Ester

The product of Preparation 11 was dissolved in TFA/DCM (25%, 52 mL) andstirred at room temperature for 2 h. The solvent was then removed underreduced pressure and the crude residue dissolved in dichloromethane (30mL) and washed with 1N sodium hydroxide (15 mL). The organic phase wasseparated, dried (magnesium sulfate) and the solvent was removed underreduced pressure to give the title compound (1.61 g, 90% yield).

Preparation 13

Biphenyl-2-ylcarbamic Acid1-[2-(4-Aminomethylbenzoylamino)ethyl]piperidin-4-yl Ester

To a stirred solution of the product of Preparation 12 (339 mg, 1 mmol),4-(tert-butoxycarbonylaminomethyl)benzoic acid (301 mg, 1.2 mmol) andHATU (456 mg, 1.2 mmol) in DMF (2 mL) was added DIPEA (226 μL, 1.3mmol). The reaction mixture was stirred at room temperature overnightand then the solvent was removed under reduced pressure. The resultingresidue was dissolved in dichloromethane (20 mL) and washed withsaturated aqueous sodium bicarbonate solution (10 mL). The organic phasewas dried (magnesium sulfate) and the solvent was removed under reducedpressure. The crude product was dissolved in TFA/DCM (25%, 10 mL) andthis mixture was stirred at room temperature for 2 h. The solvent wasremoved under reduced pressure and the crude residue was dissolved indichloromethane (15 mL) and washed with 1N sodium hydroxide (5 mL). Theorganic phase was separated, dried (magnesium sulfate) and the solventwas removed under reduced pressure to afford the title compound (472 mg,˜100% over 2 steps).

Preparation 14

Biphenyl-2-ylcarbamic Acid 1-(2-Aminoethyl)piperidin-4-yl Ester

2-tert-Butoxycarbonylaminoethyl bromide (1.22 g, 5.44 mmol) was added toa solution of the product of Example 1, Step 1 (1.46 g, 4.95 mmol) anddiisopropylethylamine (1.03 mL, 5.94 mmol) in acetonitrile (24 mL). Thereaction mixture was stirred at 65° C. for 12 hours, at which time MSanalysis showed that the reaction was completed. The reaction mixturewas concentrated to dryness and then dichloromethane (10 mL) was added.Trifluoroacetic acid was added to this mixture and the mixture wasstirred at room temperature for 4 hours, at which time MS analysisshowed that the reaction was complete. The mixture was then concentratedto half its volume and 1N sodium hydroxide was added to the solutionuntil the pH was adjusted to 14. The organic layer was washed withbrine, then dried over magnesium sulfate and filtered. The filtrate wasconcentrated to give 1.6 g of the title compound as a solid. MS m/z:[M+H⁺] calcd for C₂₀H₂₅N₃O₂ 340.2. found 340.

Preparation 15

1-[1-(9-Benzylaminononyl)piperidin-4-yl]-3-biphenyl-2-ylurea

N-Benzylamine (0.903 ml, 8.30 mmol) was added to a solution of theproduct of Preparation 2 (2.40 g, 5.52 mmol) in methanol (25 mL) and theresulting mixture was stirred at ambient temperature. After 10 min,sodium triacetoxyborohydride (1.75 g, 8.30 mmol) was added to thereaction mixture. The progress of the reaction was followed by HPLCanalysis. After 2 h at ambient temperature, the reaction was quenchedwith water (5 mL) and then concentrated to half its volume under vacuum.The reaction mixture was diluted with dichloromethane (15 mL) and washedwith 1N sodium hydroxide (2×10 mL) and then brine (5 mL). The organiclayer was dried over magnesium sulfate and concentrated to yield thetitle compound.

Preparation 16

2-Benzyloxy-5-(2-bromoacetyl)benzoic Acid Methyl Ester (a)2-Benzyloxy-5-acetylbenzoic Acid Methyl Ester

Methyl 5-acetylsalicylate (100 g, 0.515 mol) was dissolved inacetonitrile (1 L) in a 2 L flask under reflux conditions and a nitrogenatmosphere. Potassium carbonate (213.5 g, 1.545 mol) was addedportion-wise over 15 min. Benzyl bromide (67.4 mL, 0.566 mol) was addedusing a dropping funnel over 15 min. The reaction was heated to 85° C.for 9 h, and then filtered and rinsed with acetonitrile (100 mL). Thesolution was concentrated to about 300 mL volume under reduced pressureand partitioned between water (1 L) and ethyl acetate (1 L). The organiclayer was washed with saturated sodium chloride (250 mL), dried usingmagnesium sulfate (75 g), and then filtered and rinsed with ethylacetate (100 mL). The organic layer was concentrated to give2-benzyloxy-5-acetylbenzoic acid methyl ester as a solid (100% yield).

(b) 2-Benzyloxy-5-(2-bromoacetyl)benzoic Acid Methyl Ester

The product of step (a) (10.0 g, 35.2 mmol) was dissolved in chloroform(250 mL) in a 500 mL flask under a nitrogen atmosphere. Bromine (1.63mL, 31.7 mmol) dissolved in chloroform (50 mL) was added using adropping funnel over 30 min. The reaction mixture was stirred for 2.5 hand then concentrated to give a solid. The solid was dissolved intoluene (150 mL) with some gentle heat, followed by the addition ofethyl ether (150 mL) to yield the title compound as a crystalline solid(55% yield).

Preparation 17

5-[2-(Benzyl-{9-[4-(3-biphenyl-2-ylureido)piperidin-1-yl]nonyl}amino)acetyl]-2-benzyloxybenzoicAcid Methyl Ester

The product of Preparation 16 (371 mg, 1.00 mmol) was added to asolution of the product of Preparation 15 (448 mg, 0.85 mmol) indimethyl sulfoxide (4.5 mL) followed by the addition of potassiumcarbonate (234 mg, 1.7 mmol). The reaction mixture was stirred at 40° C.for 6 h, at which time the product of Preparation 15 was no longerobserved by HPLC analysis. The reaction mixture was cooled to ambienttemperature and filtered, and then diluted with ethanol (4 mL). Sodiumborohydride (63 mg, 1.7 mmol) was added to the reaction mixture and thereaction was stirred at ambient temperature for 24 h. The reactionmixture was quenched with 0.5 M ammonium chloride (5 mL) and extractedinto ethyl acetate (2×10 mL). The combined organic layers were washedwith saturated sodium bicarbonate (10 mL) and then with brine (5 mL).The organic layer was dried over magnesium sulfate and the solvent wasremoved under reduced pressure. The crude residue was purified bychromatography on silica gel (3% methanol in chloroform) to give thetitle compound.

Preparation 18

1-[1-(9-{Benzyl-[2-(4-benzyloxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}nonyl)piperidin-4-yl]-3-biphenyl-2-ylurea

A solution of the product of Preparation 17 (163 mg, 0.20 mmol) intetrahydrofuran (1.00 mL) was cooled to 0° C. Lithium aluminium hydride(1.0 M in THF; 0.50 mL, 0.50 mmol) was added dropwise to the mixture.After 1 h, the reaction mixture was quenched with water (1 mL) anddiluted with ethyl acetate (2 mL). The organic layer was washed withbrine, dried over magnesium sulfate, and the organic extracts werecombined and concentrated to give the title compound.

Preparation 19

Biphenyl-2-ylcarbamic Acid1-{2-[((1R,3S)-3-Aminocyclopentanecarbonyl)amino]-ethyl}piperidin-4-ylEster

To a stirred solution of the product of Preparation 14 (318 mg, 0.94mmol), (1R,3S)-3-tert-butoxycarbonylaminocyclopentanecarboxylic acid(258 mg, 1.1 mmol) and HATU (428 mg, 1.1 mmol) in DMF (5 mL) was addedDIPEA (245 μL, 1.09 mmol). The reaction mixture was stirred at roomtemperature overnight and then the solvent was removed under reducedpressure. The resulting residue was dissolved in dichloromethane (20 mL)and washed with saturated aqueous sodium bicarbonate solution (10 mL).The organic layer was dried (magnesium sulfate) and the solvent wasremoved under reduced pressure. The crude product was purified by flashchromatography (5-10% MeOH/DCM) and then dissolved in a trifluoroaceticacid/DCM mixture (1 mL/5 mL) and stirred at room temperature for 1 h.The solvent was removed under reduced pressure. The residue wasdissolved in dichloromethane (20 mL) and washed with 1M sodium hydroxide(10 mL), dried (magnesium sulfate) and the solvent reduced to yield thetitle compound (167 mg, 39% yield).

Preparation 20

4-(tert-Butoxycarbonylaminomethyl)-2-chlorophenylamine

A stirred solution of 4-aminomethyl-2-chlorophenylamine (940 mg, 6 mmol)and di-tert-butyl dicarbonate (1.44 g, 6.6 mmol) in dichloromethane (30mL) was stirred at room temperature for 4 h, at which time the reactionwas determined to be complete by LCMS. The reaction mixture was thenwashed with saturated aqueous sodium bicarbonate (15 mL) and the organiclayer was dried over sodium sulfate and the solvent was removed underreduced pressure. The resulting orange solid was recrystallized fromethyl acetate to give the title intermediate as a white solid (˜100%yield).

Preparation 21

N-[4-(tert-Butoxycarbonylaminomethyl)-2-chlorophenyl]acrylamide

To a stirred solution of the product of Preparation 20 (1.54 g, 6.0mmol) in a mixture of diethyl ether (35 mL) and 1 M sodium hydroxide (35mL) was added dropwise acryloyl chloride (687 μL, 8.45 mmol). After 1 h,the organic layer was separated, dried (Na₂SO₄) and the solvent wasremoved under reduced pressure to give the title intermediate as a whitesolid (1.8 g, 96% yield).

Preparation 22

Biphenyl-2-ylcarbamic Acid1-[2-(4-(tert-Butoxycarbonylaminomethyl)-2-chlorophenylcarbamoyl)ethyl]piperidin-4-ylEster

A solution of the product of Example 1, Step 1 (1.04 g, 3.5 mmol) andthe product of Preparation 21 (1.19 g, 3.85 mmol) in a mixture ofdichloromethane and methanol (12 mL, 1:1) was heated at 60° C. for 12 h.The reaction mixture was allowed to cool and the solvent was removedunder reduced pressure. The crude material was purified by columnchromatography (5-10% MeOH/DCM) to give the title intermediate as awhite solid (2.00 g, 94% yield).

Preparation 23

Biphenyl-2-ylcarbamic Acid1-[2-(4-Aminomethyl-2-chlorophenylcarbamoyl)ethyl]-piperidin-4-yl Ester

A solution of the product of Preparation 22 (2.00 g, 3.3 mmol) wasstirred in dichloromethane (24 mL) and TFA (8 mL) for 1 h and then thesolvent was removed under reduced pressure. The crude reaction mixturewas dissolved in dichloromethane (30 mL) and washed with 1 M sodiumhydroxide (2×30 mL). The organic layer was dried (Na₂SO₄) and thesolvent was removed under reduced pressure to give the titleintermediate as an oily white solid (1.46 g, 88% yield).

Preparation 24

2-Chloroethanesulfonic Acid (5-tert-Butoxycarbonylaminopentyl)amide

To a stirred solution of 5-(tert-butoxycarbonylamino)pentylamine (1.00g, 4.94 mmol) and triethylamine (689 μL g, 4.94 mmol) in dichloromethane(22 mL) at 0° C. was added 2-chloro-1-ethanesulfonyl chloride (470 μL,4.50 mmol). The reaction mixture was stirred for 2 h at room temperatureand then washed with saturated aqueous sodium bicarbonate solution (15mL). The organic layer was dried (Na₂SO₄) and the solvent was removedunder reduced pressure to give the title compound (100% yield), whichwas used in the next step without further purification.

Preparation 25

Biphenyl-2-ylcarbamic Acid1-[2-(5-tert-Butoxycarbonylaminopentylsulfamoyl)-ethyl]piperidin-4-ylEster

A solution of the product of Example 1, Step 1 (1.33 g, 3.5 mmol) andthe product of Preparation 24 (1.62 g, 4.94 mmol) in dichloromethane andmethanol (22 mL, 1:1) was heated at 60° C. for 5 h. The reaction mixturewas allowed to cool to room temperature and the solvent was removedunder reduced pressure. The crude residue was dissolved indichloromethane (20 mL) and washed with saturated aqueous sodiumbicarbonate solution (10 mL). The organic layer was then dried (Na₂SO₄)and solvent removed under reduced pressure. The crude residue waspurified by column chromatography (5-10% MeOH/DCM) to give the titleintermediate as a white solid (1.6 g, 55%). MS m/z M+H⁺=589.6.

Preparation 26

Biphenyl-2-ylcarbamic Acid1-[2-(5-Aminopentylsulfamoyl)ethyl]piperidin-4-yl Ester

A solution of the product of Preparation 25 (1.6 g, 2.72 mmol) wasstirred in dichloromethane (21 mL) and TFA (7 mL) for 1 h and then thesolvent was removed under reduced pressure. The crude reaction mixturewas dissolved in dichloromethane (30 mL) and washed with 1 M sodiumhydroxide (2×30 mL). The organic layer was dried (Na₂SO₄) and thesolvent was then removed under reduced pressure to give the titleintermediate as an oily white solid (1.19 g, 90% yield).

Preparation 27

Biphenyl-2-ylcarbamic Acid1-{2-[(4-Formylbenzenesulfonyl)methylamino]-ethyl}piperidin-4-yl Ester

To a stirred solution of the product of Preparation 8 (350 mg, 1 mmol)and triethylamine (167 μL, 1.2 mmol) in dichloromethane (5 mL) was added4-formylbenzenesulfonyl chloride (225 mg, 1.1 mmol). After 1 h at roomtemperature, the reaction was complete by MS and the reaction mixturewas then washed with saturated aqueous sodium bicarbonate solution (5mL). The organic layer was then dried (Na₂SO₄) and solvent removed underreduced pressure to give the title intermediate (323 mg, 62% yield). MSm/z M+H⁺=522.4.

Preparation 28

(3-Aminomethylphenyl)methanol Hydrochloride (a)(3-tert-Butoxycarbonylmethylphenyl)methanol

Borane dimethyl sulfide (2.05 mL, 21.6 mmol) was added to a solution of3-(tert-butoxycarbonylaminomethyl)benzoic acid (1.81 g, 7.20 mmol) intetrahydrofuran (24 mL). and the resulting mixture was stirred at roomtemperature for 3 hours. The reaction mixture was then diluted withethyl acetate (20 mL) and the layers were separated. The organic layerwas washed with saturated sodium bicarbonate, saturated sodium chloride,dried over magnesium sulfate and concentrated to give the title compoundas a yellow oil (1.71 g).

(b) (3-Aminomethylphenyl)methanol Hydrochloride

To the product of step (a) (1.71 g, 7.2 mmol) was added a solution of 4M hydrochloric acid in dioxane (9 mL, 36 mmol) and the resulting mixturewas stirred at room temperature for 1 h. The reaction mixture was thenconcentrated and the residue was diluted with diethyl ether (50 mL) andfiltered to provide the title compound as a white solid (1.09 g).

Preparation 29

Biphenyl-2-ylcarbamic Acid1-{2-[3-(3-Hydroxymethylbenzyl)ureido]ethyl}piperidin-4-yl Ester

A 0.2 M solution of the product of Preparation 12 (760 mg, 2.24 mmol) inN,N-dimethylformamide was added dropwise to a solution of1,1′-carbonyldiimidazole (364 mg, 2.24 mmol) and diisopropylethylamine(0.31 mL, 2.24 mmol) in N,N-dimethylformamide (11 mL) and the resultingmixture was stirred at room temperature for 2 h. Diisopropylethylamine(0.31 mL, 2.24 mmol) and the product of Preparation 28 (578 mg, 3.4mmol) were added and this mixture was stirred at 50° C. for 12 hours.The reaction mixture was then concentrated to dryness and the residuewas diluted with dichloromethane (20 mL) and this solution was washedwith saturated sodium bicarbonate (2×), saturated sodium chloride, driedover magnesium sulfate, and concentrated to provide the title compound(1.12 g). LCMS (2-90) R_(t)=4.01 min.; MS m/z M+H=503.5.

Preparation 30

Biphenyl-2-ylcarbamic Acid1-{2-[3-(3-Formylbenzyl)ureido]ethyl}piperidin-4-yl Ester

A solution of the product of Preparation 29 (1.12 g, 2.23 mmol) indichloromethane (11.1 mL) was cooled to 0° C. and diisopropylethylamine(1.17 mL, 6.70 mmol) and dimethyl sulfoxide (0.949 mL, 13.4 mmol) wereadded. After about 10 minutes, pyridine sulfur trioxide complex (1.06 g,6.70 mmol) was added and the resulting mixture was stirred at 0° C. for2 h. The reaction was then quenched with water (15 mL) and the organiclayer was washed with cold water (3×), dried over magnesium sulfate andconcentrated to provide the title compound as a yellow crisp (609 mg).LCMS (2-90) R_(t)=4.13 min; MS m/z M+H=501.3.

Preparation 31

Biphenyl-2-ylcarbamic Acid 1-[(E)-3-(4-Nitrophenyl)allyl]piperidin-4-ylEster

The product of Example 1, Step 1 (2.96 g, 0.01 mol) andp-nitrocinnamaldehyde (1.77 g, 0.01 mol) were stirred in 50 mL ofdichloromethane for 2 h. Sodium triacetoxyborohydride (6.33 g, 0.03 mol)was added and the resulting mixture was stirred for 2 h. The reactionwas then quenched with 10 mL of water and this mixture was diluted withdichloromethane (100 mL). The organic layer was washed with saturatedsodium bicarbonate (2×), brine, dried over Na₂SO₄, filtered andconcentrated to provide the title compound as a yellow foam (3.8 g, 80%yield).

Preparation 32

Biphenyl-2-ylcarbamic Acid 1-[3-(4-Aminophenyl)propyl]piperidin-4-ylEster

The product of Preparation 31 (2.5 g, 5.4 mmol) was dissolved in 100 mLof ethanol and the resulting solution was purged with nitrogen for 30min. Palladium on carbon (2.5 g; 50% w/w water; 10% Pd; 1.1 mmol Pd) wasthen added while degassing with nitrogen. This mixture was then placedunder hydrogen (50 psi) until hydrogen was no longer consumed (˜30minutes). The mixture was then purged with nitrogen, filtered throughCelite and concentrated. The residue was dissolved in ethyl acetate andthis mixture was washed with saturated sodium bicarbonate (2×), brine,dried (Na₂SO₄), filtered and concentrated to provide the title compound(2.08 g, 90% yield). MS m/z M+H=430.5.

Preparation 33

Biphenyl-2-ylcarbamic Acid1-[2-Fluoro-3-(4-hydroxymethylpiperidin-1-ylmethyl)-benzyl]piperidin-4-ylEster

The product of Example 1, Step 1 (500 mg, 1.69 mmol),2,6-bis(bromomethyl)-1-fluorobenzene (476 mg, 1.69 mmol,piperidin-4-ylmethanol (195 mg, 1.69 mmol) and potassium carbonate (466mg, 3.37 mmol) were suspended in acetonitrile (5 mL) and stirred at roomtemperature for 18 h. The reaction mixture was then concentrated and theresidue was dissolved in dichloromethane/water. The layers wereseparated and the organic layer was washed with water (2×), brine, dried(MgSO₄) and concentrated. The crude material was purified by silica gelcolumn chromatography eluting with 3% methanol/chloroform to give thetitle compound as a white foam (282 mg). MS m/z M+H=532.3.

Preparation 34

Biphenyl-2-ylcarbamic Acid1-[2-Fluoro-3-(4-formylpiperidin-1-ylmethyl)benzyl]-piperidin-4-yl Ester

The product of Preparation 33 (282 mg, 0.53 mmol) was dissolved indichloromethane and to this mixture was added diisopropylethylamine (280μL, 1.6 mmol) and dimethyl sulfoxide (115 μL, 1.6 mmol). The reactionmixture was cooled to −15° C. under nitrogen and pyridine sulfurtrioxide complex (255 mg, 1.6 mmol) was added and the resulting mixturewas stirred for 40 min. The reaction was then quenched with water andthe layers were separated. The organic layer was washed with aqueousNaH₂PO₄ (1M×3), brine, dried (MgSO₄) and concentrated to provide thetitle compound as a foam (253 mg). MS m/z M+H=530.4.

Preparation 35

2-[4-(3-Bromopropoxy)phenyl]ethanol

To a solution of 4-hydroxyphenethyl alcohol (4.37 g, 31.0 mmol) andpotassium carbonate (6.55 g, 47.0 mmol) in acetonitrile (62.0 mL) wasadded 1,3 dibromopropane (31.0 mL, 316 mmol). The reaction mixture washeated to 70° C. for 12 hours and then cooled to room temperature,filtered and concentrated under vacuum. The resulting oil was purifiedby silica gel chromatography using a mixture of 4:1 hexanes and ethylacetate to give the title compound (6.21 g) as a white solid.

Preparation 36

Biphenyl-2-ylcarbamic Acid1-{3-[4-(2-Hydroxyethyl)phenoxy]propyl}piperidin-4-yl Ester

To a solution of the product of Preparation 35 (1.11 g, 4.30 mmol) anddiisopropylethylamine (0.90 mL, 5.10 mmol) in acetonitrile (21.5 mL) wasadded the product of Example 1, Step 1 (1.27 g, 4.30 mmol) and theresulting mixture was stirred at 60° C. for 12 h. The reaction mixturewas then diluted with dichloromethane (20 mL) and washed with saturatedsodium bicarbonate (25 mL), saturated sodium chloride (25 mL), driedover magnesium sulfate and concentrated to provide the title compound(1.98 g, 85% purity). MS m/z M+H=475.5.

Preparation 37

Biphenyl-2-ylcarbamic Acid1-{3-[4-(2-Oxoethyl)phenoxy]propyl}piperidin-4-yl Ester

A solution of the product of Preparation 36 (723 mg, 1.53 mmol) anddichloromethane (75 mL) was cooled to about 5° C. anddiisopropylethylamine (798 mL, 4.58 mmol) and dimethyl sulfoxide (649mL, 9.15 mmol) were added. Pyridine sulfur trioxide (728 mg, 4.58 mmol)was then added and the resulting mixture was stirred at 5° C. for 45min. The reaction mixture was then diluted with dichloromethane (20 mL)and washed with saturated sodium bicarbonate (25 mL), saturated sodiumchloride (25 mL), dried over magnesium sulfate and concentrated toprovide the title compound (604 mg). MS m/z M+H=473.4.

Preparation 38

Methyl 4-Iodophenylacetate

To a stirred solution of 4-iodophenylacetic acid (5.0 g, 19.1 mmol) inMeOH (200 mL) was added 4N hydrochloric acid in dioxane (10 mL). Thereaction mixture was stirred for 24 h at room temperature and then thesolvent was removed under reduced pressure to give the title compound(5.17 g, 98% yield), which was used without further purification.

Preparation 39

Methyl [4-(4-Hydroxybut-1-ynyl)phenyl]acetate

To a stirred solution of the product of Preparation 38 (4.5 g, 16.3mmol) in diethylamine (100 mL) was added but-3-yn-1-ol (1.9 mL, 32.6mmol), Pd(PPh₃)₂Cl₂ (500 mg, 1.63 mmol) and CuI (154 mg, 0.815 mmol) andresulting mixture was stirred for 17 h at room temperature. The solventwas then removed under reduced pressure and the residue was dissolved indiethyl ether (200 mL) and this solution was filtered to remove salts.The solvent was then removed under reduced pressure and the crudeproduct was purified by silica gel chromatography (60% EtOAc/Hexane) toafford the title intermediate (3.03 g, 91% yield).

Preparation 40

Methyl [4-(4-Hydroxybutyl)phenyl]acetate

A stirred solution of the product of Preparation 39 (2.8 g, 12.8 mmol)in methanol (50 mL) was flushed with nitrogen and then 10% palladium oncarbon (400 mg, 20% wt/wt) was added. The reaction flask was thenalternately placed under vacuum and flushed with hydrogen for cycles andthen stirred under hydrogen for 14 h. The reaction mixture was flushedwith nitrogen and then filtered and the solvent removed under reducedpressure to give the title compound (2.75 g, 97% yield), which was usedwithout further purification.

Preparation 41

Methyl(4-{4-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]butyl}phenyl)acetate(a) Methyl {4-[4-(Toluene-4-sulfonyloxy)butyl]phenyl}acetate

To a stirred solution of the product of Preparation 40 (2.6 g, 12.5mmol) in THF (100 mL) was added DABCO (2.6 g, 25.0 mmol) and thenp-toluenesulfonyl chloride (2.44 g, 13.75 mmol). The reaction mixturewas stirred at room temperature for 23 h and then solvent was removedunder reduced pressure and the residue was dissolved in dichloromethane(200 mL). The organic layer was then washed with water (2×100 mL), 1Nhydrochloric acid (100 mL), aqueous saturated sodium chloride solution(100 mL), dried (MgSO₄), filtered and the solvent removed under reducedpressure to give the title compound, which was used without furtherpurification.

(b) Methyl(4-{4-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]butyl}phenyl)acetate

To the crude product from step (a) was added DMF (50 mL),diisopropylethylamine (3.0 mL, 17.3 mmol) and the product of Preparation8 (2.4 g, 8.1 mmol). The reaction mixture was stirred at roomtemperature for 18 h and then the solvent was removed under reducedpressure to give the title compound (3.5 g, 86.3% yield). MS m/z 501.6(MH⁺), R_(f) 4.89 min (10-70% ACN:H₂O, reverse phase HPLC).

Preparation 42

Biphenyl-2-ylcarbamic Acid1-{4-[4-(2-Hydroxyethyl)phenyl]butyl}piperidin-4-yl Ester

To a stirred solution of the product of Preparation 41 (2.0 g, 4.0 mmol)in THF (100 mL) was added dropwise DIBAL (24 mL, 24 mmol, 1.0 M in THF).After the addition was complete, the reaction mixture was stirred for 3h and then quenched by slow addition of methanol (until gas evolutionceased). The mixture was then stirred for 30 min. and then ethyl acetate(200 mL) and aqueous 1N sodium hydroxide (200 mL) were added. Theorganic layer was separated and washed with aqueous saturated sodiumchloride solution (100 mL), dried (MgSO₄), filtered and the solventremoved under reduced pressure to give the title compound (1.3 g, 69%yield), which was used without further purification. MS m/z 473.4 (MH⁺),R_(f) 4.53 min (10-70% ACN:H₂O, reverse phase HPLC).

Preparation 43

Ethyl 3-[5-(2-Ethoxycarbonylvinyl)thiophen-2-yl]acrylate

To a stirred solution of sodium hydride (2.1 g, 53 mmol, 60% in mineraloil) in THF (200 mL) was slowly added triethylphosphonoacetate (10 mL,50 mmol) Hydrogen gas evolution was observed and the reaction wasstirred until gas evolution ceased (about 30 min). To this reactionmixture was added 2,5-thiophenedicarboxaldehyde (3 g, 21 mmol) and thereaction mixture was stirred for 1 h. The solvent was removed underreduced pressure and the residue was dissolved in dichloromethane (200mL). The organic layer was washed with water (100 mL), aqueous 1Nhydrochloric acid (100 mL), aqueous saturated sodium chloride solution(100 mL), dried (MgSO₄), filtered and the solvent removed under reducedpressure to give the title compound (5.8 g, 98% yield), which was usedwithout further purification.

Preparation 44

Ethyl 3-[5-(2-Ethoxycarbonylethyl)thiophen-2-yl]propionate

A stirred solution of the product of Preparation 43 (5.8 g, 21 mmol) inmethanol (200 mL) was flushed with nitrogen and 10% palladium on carbon(576 mg, 10% wt/wt) was added. The reaction flask was alternately placedunder vacuum and flushed with hydrogen for 3 cycles and then thereaction mixture was stirred under hydrogen for 1 h. The mixture wasthen was flushed with nitrogen, filtered and the solvent removed underreduced pressure to give the title compound (5.8 g, 99% yield), whichwas used without further purification.

Preparation 45

3-[5-(3-Hydroxypropyl)thiophen-2-yl]propan-1-ol

To a stirred solution of DIBAL (88 mL, 88 mmol, 11.0M in cyclohexane) inTHF (300 mL) at −78° C. was added dropwise the product of Preparation 44(5.0 g, 17.6 mmol). After the addition was complete, the reactionmixture was warmed to room temperature over 30 min and then quenched byslow addition of aqueous 1N hydrochloric acid (200 mL). Dichloromethane(400 mL) was added and the layers were separated. The aqueous layer waswashed with dichloromethane (4×100 mL) and the combined organic layerswere washed with aqueous saturated sodium chloride solution (100 mL),dried (MgSO₄), filtered and the solvent removed under reduced pressureto give the title compound (3.0 g, 85% yield), which was used withoutfurther purification.

Preparation 46

Biphenyl-2-ylcarbamic Acid1-{3-[5-(3-Hydroxypropyl)thiophen-2-yl]propyl}piperidin-4-yl Ester (a)Toluene-4-sulfonic Acid 3-[5-(3-Hydroxypropyl)thiophen-2-yl]propyl Ester

To a stirred solution of the product of Preparation 45 (423 mg, 2.1mmol) in THF (20 mL) was added DABCO (420 mg, 4.2 mmol) and thenp-toluenesulfonyl chloride (442 mg, 2.3 mmol). The reaction mixture wasstirred at room temperature for 2 h and then the solvent was removedunder reduced pressure and the residue was dissolved in dichloromethane(200 mL). The organic layer was washed with water (2×100 mL), aqueoussaturated sodium chloride solution (100 mL), dried MgSO₄), filtered andthe solvent removed under reduced pressure to give the title compound,which was used without further purification.

(b) Biphenyl-2-ylcarbamic Acid1-{3-[5-(3-Hydroxypropyl)thiophen-2-yl]propyl}piperidin-4-yl Ester

To the product from step (a) was added acetonitrile (20 mL),diisopropylethylamine (0.5 mL, 2.8 mmol) and the product of Example 1,Step 1 (626 mg, 2.11 mmol). The reaction mixture was heated to 50° C.for 20 h and then cooled to room temperature and the solvent was removedunder reduced pressure. The residue was purified by silica gelchromatography (5% MeOH/DCM with 0.6% NH₃ (aq)) to afford the titlecompound (450 mg, 44% yield). MS m/z (MH⁺) 479.6; R_(f) 4.15 min (10-70%ACN:H₂O, reverse phase HPLC).

Preparation 47

Methyl 4-Amino-5-chloro-2-methoxybenzoate

To a solution of 4-amino-5-chloro-2-methoxybenzoic acid (1.008 g, 5.0mmol) in a mixture of toluene (9 mL) and methanol (1 mL) at 0° C. wasadded (trimethylsilyl)diazomethane (2.0 M in hexane, 3.0 mL, 6.0 mmol)dropwise. The reaction mixture was then warmed to room temperature andstirred for 16 h. Excess (trimethylsilyl)diazomethane was quenched byadding acetic acid until the bright yellow color of the reaction mixturedisappeared. The mixture was then concentrated in vacuo to give thetitle compound as an off-white solid, which was used without furtherpurification.

Preparation 48

Methyl 4-Acryloylamino-5-chloro-2-methoxybenzoate

To crude product of Preparation 47 was added dichloromethane (10 mL, 0.5M) and triethylamine (2.1 mL, 15 mmol). This mixture was cooled to 0° C.and acryloyl chloride (812 μL, 10 mmol) was added dropwise withstirring. After 2 h, the reaction was quenched by adding methanol (about2 mL) at 0° C. and the resulting mixture was stirred at room temperaturefor 15 min and then concentrated in vacuo. Dichloromethane (30 mL) andwater (30 mL) were added to the residue and this mixture was mixedthoroughly. The layers were separated and the aqueous layer wasextracted with dichloromethane (20 mL). The organic layers werecombined, dried (Na₂SO₄), filtered and the solvent was removed in vacuoto give the title compound as a brown foamy solid, which was usedwithout further purification.

Preparation 49

Methyl4-{3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-5-chloro-2-methoxybenzoate

To the crude product from Preparation 48 was added the product ofExample 1, Step 1 (1.33 g, 4.5 mmol) and a mixture of THF (22.5 mL) andmethanol (2.5 mL). This mixture was heated at 50° C. with stirring for16 h and then the solvent was removed in vacuo. The residue waschromatographed (silica gel; EtOAc) to give the title compound (0.82 g;R_(f)=0.4, 29% yield over 3 steps) as an off-white foamy solid. MS m/z566.4 (M+H, expected 565.20 for C₃₀H₃₂ClN₃O₆).

Preparation 50

Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-hydroxymethyl-5-methoxy-phenylcarbamoyl)ethyl]piperidin-4-ylEster

To a solution of the product of Preparation 49 (0.82 mg, 1.45 mmol) in amixture of THF (4.5 mL) and methanol (0.5 mL) at 0° C. was added lithiumborohydride (32 mg, 1.45 mmol). The reaction mixture was allowed to warmto room temperature and was stirred for 41 h. The reaction was thenquenched by adding 1N aqueous hydrochloric acid at 0° C. until no morebubbling was observed and this mixture was stirred for 10 min. Thesolvent was removed in vacuo and the residue was dissolved inacetonitrile (about 2 mL). This solution was purified by prep-RP-HPLC(gradient: 2 to 50% acetonitrile in water with 0.05% TFA). Theappropriate fractions were collected and combined and lyophilized togive the title compound as a trifluoroacetate salt. This salt wastreated with isopropyl acetate (10 mL) and 1N aqueous sodium hydroxide(10 mL) and the organic layer was collected, dried (Na₂SO₄), filteredand the solvent was removed in vacuo to give the title compound (161 mg,21% yield) as a white foamy solid. MS m/z 538.4 (M+H, expected 537.20for C₂₉H₃₂ClN₃O₅).

Preparation 51

Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-formyl-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylEster

To a solution of the product of Preparation 50 (161 mg, 0.3 mmol) indichloromethane (3 mL) was added dimethyl sulfoxide (213 μL, 3.0 mmol)and diisopropylethylamine (261 μL, 1.5 mmol). This mixture was cooled to−20° C. and sulfur trioxide pyridine complex (238 mg, 1.5 mmol) wasadded slowly. After 30 min, the reaction mixture was quenched by addingwater (about 3 mL). The layers were separated and the organic layer wasdried (Na₂SO₄), filtered and the solvent was removed in vacuo to givethe title compound as a light yellow solid. MS m/z 536.3 (M+H, expected535.19 for C₂₉H₃₀ClN₃O₅).

Preparation 52

Biphenyl-2-ylcarbamic Acid1-[2-(4-[1,3]dioxolan-2-ylphenylcarbamoyl)-ethyl]-4-methylpiperidin-4-ylEster

A mixture of biphenyl-2-ylcarbamic acid 4-methylpiperidin-4-yl ester(2.73 g, 8.79 mmol) and N-(4-[1,3]dioxolan-2-yl-phenyl)acrylamide (2.05g, 8.80 mmol) were heated in 100 mL of 1:1 methanol/dichloromethane at50° C. under nitrogen for 1 h. The solution was then diluted with ethylacetate and the organic layer was washed with water, brine, dried(MgSO₄) and concentrated under reduced pressure to give the titlecompound. MS m/z calcd for C₃₁H₃₅N₃O₅ (M+H)⁺ 530.6. found 530.4.

Preparation 53

Biphenyl-2-ylcarbamic Acid1-[2-(4-Formylphenylcarbamoyl)ethyl]-4-methylpiperidin-4-yl Ester

The product of Preparation 52 was redissolved in 40 mL of methanol and25 mL of aqueous 1 N hydrochloric acid was added. The resulting mixturewas stirred at room temperature overnight and the organic solvent wasremoved under reduced pressure. The residue was dissolved in ethylacetate and the organic layer was washed with water, brine, dried(MgSO₄) and the solvent removed under reduced pressure. The product wastriturated with dichloromethane to give the title compound as a whitepowder (2.47 g). LCMS (2-90) R_(t)=4.27 min; MS m/z calcd for C₂₉H₃₁N₃O₄(M+H)⁺ 486.6. found 486.5.

Preparation 54

Biphenyl-2-ylcarbamic acid (R)-(1-azabicyclo[3.2.1]oct-4-yl) Ester

2-Biphenyl isocyanate (1.00 g, 5.12 mmol) and (R)-(−)-3-quinuclidinolhydrochloride (921 mg, 5.63 mmol) were heated together inN,N-dimethylformamide (2.06 mL) at 110° C. for 12 h. The reactionmixture was cooled and diluted with ethyl acetate (15 mL) and thenwashed with saturated aqueous sodium bicarbonate (2×10 mL). The organiclayer was extracted with 1 M hydrochloric acid (3×20 mL) and thecombined aqueous extracts were made basic to pH 8-9 with potassiumcarbonate. The aqueous layer was then extracted with ethyl acetate (3×20mL) and the combined organic layers were dried (magnesium sulfate) andsolvent was removed under reduced pressure to give the title compound asa yellow oil (1.64 g, 99% yield).

Preparation 55

(R)-4-(Biphenyl-2-ylcarbamoyloxy)-1-(9-bromononyl)-1-azoniabicyclo[3.2.1]octaneBromide

To a stirred solution of the product of Preparation 54 (1.21 g, 3.76mmol) and triethylamine (1.05 mL, 7.52 mmol) in acetonitrile (18.8 mL)was added 1,9-dibromononane (994 μL, 4.89 mmol) and the reaction mixturewas heated at 50° C. for 4 h. The reaction mixture was then cooled andthe solvent was removed under reduced pressure. The residue wasdissolved in dichloromethane (20 mL) and the organic layer was washedwith saturated aqueous sodium bicarbonate (10 mL), dried (magnesiumsulfate) and solvent removed under reduced pressure. The crude productwas purified by flash chromatography (10% methanol/dichloromethane, 0.5%ammonium hydroxide) to give the title compound (1.04 g, 1.97 mmol, 52%yield).

Preparation 56

(R)-1-(9-N,N-Di(tert-butoxycarbonyl)aminononyl)-4-(biphenyl-2-ylcarbamoyloxy)-1-azoniabicyclo[3.2.1]octaneBromide

To a stirred solution of sodium hydride (60% dispersion in mineral oil)(126 mg, 3.15 mmol) in N,N-dimethylformamide (10 mL) under an atmosphereof nitrogen at 0° C., was added di-tert-butyl iminodicarboxylate (513mg, 2.36 mmol) in N,N-dimethylformamide (5 mL). The reaction mixture wasstirred at room temperature for 15 min and then it was cooled to 0° C.and the product of Preparation 55 (1.04 g, 1.97 mmol) inN,N-dimethylformamide (5 mL) was added. The reaction mixture was allowedto warm to room temperature over a 12 h period and then the solvent wasremoved under reduced pressure to give the title compound, which wasused without further purification.

Preparation 57

(R)-1-(9-Aminononyl)-4-(biphenyl-2-ylcarbamoyloxy)-1-azoniabicyclo[3.2.1]octaneBromide

The product of Preparation 56 (1.31 g, 1.97 mmol) was dissolved indichloromethane (15 mL) and trifluoroacetic acid (5 mL) was addedslowly. The reaction mixture was stirred at room temperature for 1 h andthen the solvent was removed under reduced pressure. The residue wasdissolved in dichloromethane (20 mL) and washed with aqueous 1 M sodiumhydroxide (20 mL). The organic layer was extracted with 1 M hydrochloricacid (3×20 mL) and the combined aqueous extracts were made basic withpotassium carbonate and extracted with dichloromethane (3×20 mL). Thecombined organic layers were dried (magnesium sulfate) and solvent wasremoved under reduced pressure to give the title compound (210 mg, 23%yield over 2 steps).

Preparation A

Cell Culture and Membrane Preparation From Cells Expressing Human β₁, β₂or β₃ Adrenergic Receptors

Chinese hamster ovarian (CHO) cell lines stably expressing cloned humanβ₁, β₂ or β₃ adrenergic receptors, respectively, were grown to nearconfluency in Hams F-12 media with 10% FBS in the presence of 500 μg/mLGeneticin. The cell monolayer was lifted with 2 mM EDTA in PBS. Cellswere pelleted by centrifugation at 1,000 rpm, and cell pellets wereeither stored frozen at −80° C. or membranes were prepared immediatelyfor use. For preparation of β₁ and β₂ receptor expressing membranes,cell pellets were re-suspended in lysis buffer (10 mM HEPES/HCl, 10 mMEDTA, pH 7.4 at 4° C.) and homogenized using a tight-fitting Dounceglass homogenizer (30 strokes) on ice. For the more protease-sensitiveβ₃ receptor expressing membranes, cell pellets were homogenated in lysisbuffer (10 mM Tris/HCl, pH 7.4) supplemented with one tablet of“Complete Protease Inhibitor Cocktail Tablets with 2 mM EDTA” per 50 mLbuffer (Roche Catalog No. 1697498, Roche Molecular Biochemicals,Indianapolis, Ind.). The homogenate was centrifuged at 20,000×g, and theresulting pellet was washed once with lysis buffer by re-suspension andcentrifugation as above. The final pellet was then re-suspended inice-cold binding assay buffer (75 mM Tris/HCl pH 7.4, 12.5 mM MgCl₂, 1mM EDTA). The protein concentration of the membrane suspension wasdetermined by the methods described in Lowry et al., 1951, Journal ofBiological Chemistry, 193, 265; and Bradford, Analytical Biochemistry,1976, 72, 248-54. All membranes were stored frozen in aliquots at −80°C. or used immediately.

Preparation B

Cell Culture and Membrane Preparation from Cells Expressing Human M₁,M₂, M₃ and M₄ Muscarinic Receptors

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in HAM's F-12 media supplemented with 10% FBS and 250 μg/mLGeneticin. The cells were grown in a 5% CO₂, 37° C. incubator and liftedwith 2 mM EDTA in dPBS. Cells were collected by 5 minute centrifugationat 650×g, and cell pellets were either stored frozen at −80° C. ormembranes were prepared immediately for use. For membrane preparation,cell pellets were resuspended in lysis buffer and homogenized with aPolytron PT-2100 tissue disrupter (Kinematica AG; 20 seconds×2 bursts).Crude membranes were centrifuged at 40,000×g for 15 minutes at 4° C. Themembrane pellet was then resuspended with re-suspension buffer andhomogenized again with the Polytron tissue disrupter. The proteinconcentration of the membrane suspension was determined by the methoddescribed in Lowry et al., 1951, Journal of Biochemistry, 193, 265. Allmembranes were stored frozen in aliquots at −80° C. or used immediately.Aliquots of prepared hM₅ receptor membranes were purchased directly fromPerkin Elmer and stored at −80° C. until use.

Assay Test Procedure A

Radioligand Binding Assay for Human β₁, β₂ and β₃ Adrenergic Receptors

Binding assays were performed in 96-well microtiter plates in a totalassay volume of 100 μL with 10-15 μg of membrane protein containing thehuman β₁, β₂ or β₃ adrenergic receptors in assay buffer (75 mM Tris/HClpH 7.4 at 25° C., 12.5 mM MgCl₂, 1 mM EDTA, 0.2% BSA). Saturationbinding studies for determination of K_(d) values of the radioligandwere done using [³H]-dihydroalprenolol (NET-720, 100 Ci/mmol,PerkinElmer Life Sciences Inc., Boston, Mass.) for the β₁ and β₂receptors and [¹²⁵I]-(−)-iodocyanopindolol (NEX-189, 220 Ci/mmol,PerkinElmer Life Sciences Inc., Boston, Mass.) at 10 or 11 differentconcentrations ranging from 0.01 nM to 20 nM. Displacement assays fordetermination of K_(i) values of test compounds were done with[³H]-dihydroalprenolol at 1 nM and [¹²⁵I]-(−)-iodocyanopindolol at 0.5nM for 10 or 11 different concentrations of test compound ranging from10 pM to 10 μM. Non-specific binding was determined in the presence of10 μM propranolol. Assays were incubated for 1 hour at 37° C., and thenbinding reactions were terminated by rapid filtration over GF/B for theβ₁ and β₂ receptors or GF/C glass fiber filter plates for the 3receptors (Packard BioScience Co., Meriden, Conn.) presoaked in 0.3%polyethyleneimine. Filter plates were washed three times with filtrationbuffer (75 mM Tris/HCl pH 7.4 at 4° C., 12.5 mM MgCl₂, 1 mM EDTA) toremove unbound radioactivity. The plates were then dried and 50 μL ofMicroscint-20 liquid scintillation fluid (Packard BioScience Co.,Meriden, Conn.) was added and plates were counted in a Packard Topcountliquid scintillation counter (Packard BioScience Co., Meriden, Conn.).Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The curveminimum was fixed to the value for nonspecific binding, as determined inthe presence of 10 μM propranolol. K_(i) values for test compounds werecalculated from observed IC₅₀ values and the K_(d) value of theradioligand using the Cheng-Prusoff equation (Cheng Y, and Prusoff W H.,Biochemical Pharmacology, 1973, 22, 23, 3099-108).

In this assay, a lower K_(i) value indicates that a test compound has ahigher binding affinity for the receptor tested. Exemplified compound ofthis invention that were tested in this assay typically were found tohave a K_(i) value of less than about 300 nM for the β₂ adrenergicreceptor. For example, the compounds of Examples 1 to 3 were found tohave K_(i) values of less than 30 nM.

Assay Test Procedure B

Radioligand Binding Assay for Muscarinic Receptors

Radioligand binding assays for cloned human muscarinic receptors wereperformed in 96-well microtiter plates in a total assay volume of 100μL. CHO cell membranes stably expressing either the hM₁, hM₂, hM₃, hM₄or hM₅ muscarinic subtype were diluted in assay buffer to the followingspecific target protein concentrations (μg/well): 10 μg for hM₁, 10-15μg for hM₂, 10-20 μg for hM₃, 10-20 μg for hM₄, and 10-12 μg for hM₅ toget similar signals (cpm). The membranes were briefly homogenized usinga Polytron tissue disruptor (10 seconds) prior to assay plate addition.Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]—NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]—NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 400 μM in dilution buffer and then serially diluted5× with dilution buffer to final concentrations ranging from 10 pM to100 μM. The addition order and volumes to the assay plates were asfollows: 25 μL radioligand, 25 μL diluted test compound, and 50 μLmembranes. Assay plates were incubated for 60 minutes at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (PerkinElmer Inc., Wellesley, Mass.) pre-treated in 1%BSA. Filter plates were rinsed three times with wash buffer (10 mMHEPES) to remove unbound radioactivity. The plates were then air driedand 50 μL Microscint-20 liquid scintillation fluid (PerkinElmer Inc.,Wellesley, Mass.) was added to each well. The plates were then countedin a PerkinElmer Topcount liquid scintillation counter (PerkinElmerInc., Wellesley, Mass.). Binding data were analyzed by nonlinearregression analysis with the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.) using the one-site competition model.K_(i) values for test compounds were calculated from observed IC₅₀values and the K_(D) value of the radioligand using the Cheng-Prusoffequation (Cheng Y; Prusoff W H. (1973) Biochemical Pharmacology,22(23):3099-108). K_(i) values were converted to pK_(i) values todetermine the geometric mean and 95% confidence intervals. These summarystatistics were then converted back to K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. Exemplified compoundof this invention that were tested in this assay typically were found tohave a K_(i) value of less than about 300 nM for the M₃ muscarinicreceptor. For example, the compounds of Examples 1 to 4 were found tohave K_(i) values of less than 10 nM.

Assay Test Procedure C

Whole-Cell cAMP Flashplate Assay in CHO Cell Lines HeterologouslyExpressing Human β₁, β₂ or β₃ Adrenergic Receptors

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with [¹²⁵I]-cAMP(NEN SMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), accordingto the manufacturers instructions. For the determination of β receptoragonist potency (EC₅₀), CHO-K1 cell lines stably expressing cloned humanβ₁, β₂ or β₃ adrenergic receptors were grown to near confluency in HAM'sF-12 media supplemented with 10% FBS and Geneticin (250 μg/mL). Cellswere rinsed with PBS and detached in dPBS (Dulbecco's Phosphate BufferedSaline, without CaCl₂ and MgCl₂) containing 2 mM EDTA or Trypsin-EDTAsolution (0.05% trypsin/0.53 mM EDTA). After counting cells in Coultercell counter, cells were pelleted by centrifugation at 1,000 rpm andre-suspended in stimulation buffer containing IBMX (PerkinElmer Kit)pre-warmed to room temperature to a concentration of 1.6×10⁶ to 2.8×10⁶cells/mL. About 60,000 to 80,000 cells per well were used in this assay.Test compounds (10 mM in DMSO) were diluted into PBS containing 0.1% BSAin Beckman Biomek-2000 and tested at 11 different concentrations rangingfrom 100 μM to 1 pM. Reactions were incubated for 10 min at 37° C. andstopped by adding 100 μL of cold detection buffer containing [¹²⁵I]-cAMP(NEN SMP004, PerkinElmer Life Sciences, Boston, Mass.). The amount ofcAMP produced (pmol/well) was calculated based on the counts observedfor the samples and cAMP standards as described in the manufacturer'suser manual. Data were analyzed by nonlinear regression analysis withthe GraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) with the sigmoidal equation. The Cheng-Prusoff equation (ChengY, and Prusoff W H., Biochemical Pharmacology, 1973, 22, 23, 3099-108)was used to calculate the EC50 values.

In this assay, a lower EC₅₀ value indicates that the test compound has ahigher functional activity at the receptor tested. Exemplified compoundof this invention that were tested in this assay typically were found tohave a EC₅₀ value of less than about 300 mM for the β₂ adrenergicreceptor. For example, the compounds of Examples 1 to 4 were found tohave EC₅₀ values of less than 30 nM.

If desired, the receptor subtype selectivity for a test compound can becalculated as the ratio of EC₅₀(β₁)/EC₅₀(β₂) or EC₅₀(β₃)/EC₅₀(β₂).Typically, compounds of this invention demonstrated greater functionalactivity at the β₂ adrenergic receptor compared to the β₁ or β₃adrenergic receptor, i.e. EC₅₀(β₁) or EC₅₀(β₃) is typically greater thanEC₅₀(β₂). Generally, compounds having selectivity for the β₂ adrenergicreceptor over the β₁ or β₃ adrenergic receptors are preferred;especially compounds having a selectivity greater than about 5; and inparticular, greater than about 10. By way of example, the compounds ofExamples 1 to 4 had ratios of EC₅₀(β₁)/EC₅₀(β₂) greater than 10.

Assay Test Procedure D

Functional Assays of Antagonism for Muscarinic Receptor Subtypes

A. Blockade of Agonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound is determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor. cAMP assays are performed in aradioimmunoassay format using the Flashplate Adenylyl Cyclase ActivationAssay System with ¹²⁵I-cAMP (NEN SMP004B, PerkinElmer Life SciencesInc., Boston, Mass.), according to the manufacturer's instructions.Cells are rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in the Cell Culture andMembrane Preparation section above. The detached cells are washed twiceby centrifugation at 650×g for five minutes in 50 mL dPBS. The cellpellet is then re-suspended in 10 mL dPBS, and the cells are countedwith a Coulter ZI Dual Particle Counter (Beckman Coulter, Fullerton,Calif.). The cells are centrifuged again at 650×g for five minutes andre-suspended in stimulation buffer to an assay concentration of1.6×10⁶-2.8×10⁶ cells/mL.

The test compound is initially dissolved to a concentration of 400 μM indilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.1 nM. Oxotremorine is diluted in a similarmanner.

To measure oxotremorine inhibition of adenylyl cyclase (AC) activity, 25μL forskolin (25 μM final concentration diluted in dPBS), 25 μL dilutedoxotremorine, and 50 μL cells are added to agonist assay wells. Tomeasure the ability of a test compound to block oxotremorine-inhibitedAC activity, 25 μL forskolin and oxotremorine (25 μM and 5 μM finalconcentrations, respectively, diluted in dPBS), 25 μL diluted testcompound, and 50 μL cells are added to remaining assay wells.

Reactions are incubated for 10 minutes at 37° C. and stopped by additionof 100 μL ice-cold detection buffer. Plates are sealed, incubatedovernight at room temperature and counted the next morning on aPerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) is calculatedbased on the counts observed for the samples and cAMP standards, asdescribed in the manufacturer's user manual. Data is analyzed bynonlinear regression analysis with the GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.) using the non-linearregression, one-site competition equation. The Cheng-Prusoff equation isused to calculate the K_(i), using the EC₅₀ of the oxotremorineconcentration-response curve and the oxotremorine assay concentration asthe K_(D) and [L], respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Exemplifiedcompounds of this invention are expected to have a K_(i) value of lessthan about 300 nM for blockade of oxotremorine-inhibition offorskolin-mediated cAMP accumulation in CHO-K1 cells expressing the hM₂receptor.

B. Blockade of Agonist-Mediated [³⁵S]GTPγS Binding

In a second functional assay, the functional potency of test compoundsis determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes were thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes were briefly homogenized using a PolytronPT-2100 tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine wasdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following was added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine (EC₉₀) and GDP (3 uM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates were then incubated at 37° C. for 60 minutes. The assayplates were filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates were rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) was added to each well, and each plate wassealed and radioactivity counted on a Topcounter (PerkinElmer). Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Exemplifiedcompound of this invention that were tested in this assay typically werefound to have a K_(i) value of less than about 300 nM for blockade ofoxotremorine-stimulated [³⁵S]GTPγS-binding in CHO-K1 cells expressingthe hM₂ receptor. For example, the compound of Example 1 was found tohave a K_(i) value of less than 10 nM.

C. Blockade of Agonist-Mediated Calcium Release Via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency can be determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors are seeded into 96-well FLIPR plates thenight before the assay is done. Seeded cells are washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in Hank's Buffered SaltSolution (HBSS) without calcium and magnesium) to remove growth mediaand leaving 50 μL/well of FLIPR buffer. The cells are then incubatedwith 50 μL/well of 4 μM FLUO-4AM (a 2× solution was made) for 40 minutesat 37° C., 5% carbon dioxide. Following the dye incubation period, cellsare washed two times with FLIPR buffer, leaving a final volume of 50μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine is first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells are first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine isgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100−F)^1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)is prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine is added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR are: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline is determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measured thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence is expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data is analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values aredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Exemplifiedcompound of this invention are expected to have a K_(i) value of lessthan about 300 nM for blockade of agonist-mediated calcium release inCHO cells stably expressing the hM₁, hM₃ and cM₅ receptors.

Assay Test Procedure E

Whole-Cell cAMP Flashplate Assay with a Lung Epithelial Cell LineEndogenously Expressing Human β₂ Adrenergic Receptor

For the determination of agonist potencies and efficacies (intrinsicactivities) in a cell line expressing endogenous levels of the β₂adrenergic receptor, a human lung epithelial cell line (BEAS-2B) wasused (ATCC CRL-9609, American Type Culture Collection, Manassas, Va.)(January B, et al., British Journal of Pharmacology, 1998, 123, 4,701-11). Cells were grown to 75-90% confluency in complete, serum-freemedium (LHC-9 MEDIUM containing Epinephrine and Retinoic Acid, cat#181-500, Biosource International, Camarillo, Calif.). The day beforethe assay, medium was switched to LHC-8 (no epinephrine or retinoicacid, cat #141-500, Biosource International, Camarillo, Calif.). cAMPassays were performed in a radioimmunoassay format using the FlashplateAdenylyl Cyclase Activation Assay System with [¹²⁵I]-cAMP (NEN SMP004,PerkinElmer Life Sciences Inc., Boston, Mass.), according to themanufacturers instructions. On the day of the assay, cells were rinsedwith PBS, lifted by scraping with 5 mM EDTA in PBS, and counted. Cellswere pelleted by centrifugation at 1,000 rpm and re-suspended instimulation buffer pre-warmed to 37° C. at a final concentration of600,000 cells/mL. Cells were used at a final concentration of 100,000 to120,000 cells/well in this assay. Test compounds were serially dilutedinto assay buffer (75 mM Tris/HCl pH 7.4 at 25° C., 12.5 mM MgCl₂, 1 mMEDTA, 0.2% BSA) in Beckman Biomek-2000. Test compounds were tested inthe assay at 11 different concentrations, ranging from 10 μM to 10 pM.Reactions were incubated for 10 min at 37° C. and stopped by addition of100 μL of ice-cold detection buffer. Plates were sealed, incubated overnight at 4° C. and counted the next morning in a Topcount scintillationcounter (Packard BioScience Co., Meriden, Conn.). The amount of cAMPproduced per mL of reaction was calculated based on the counts observedfor samples and cAMP standards, as described in the manufacturer's usermanual. Data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 4-parameter model for sigmoidal dose-response.

In this assay, a lower EC₅₀ value indicates that the test compound has ahigher functional activity at the receptor tested. Exemplified compoundof this invention that were tested in this assay typically were found tohave a EC₅₀ value of less than about 300 nM for the β₂ adrenergicreceptor. For example, the compounds of Examples 1 to 3 were found tohave EC₅₀ values ranging from less than 10 nM to less than 200 nM.

Assay Test Procedure F

Duration of Bronchoprotection in Guinea Pig Models ofAcetylcholine-Induced or Histamine-Induced Bronchoconstriction

These in vivo assays are used to assess the bronchoprotective effects oftest compounds exhibiting both muscarinic receptor antagonist and β₂adrenergic receptor agonist activity. To isolate muscarinic antagonistactivity in the acetylcholine-induced bronchoconstriction model, theanimals are administered propanolol, a compound that blocks β receptoractivity, prior to the administration of acetylcholine. Duration ofbronchoprotection in the histamine-induced bronchoconstriction modelreflects β₂ adrenergic receptor agonist activity.

Groups of 6 male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g are individuallyidentified by cage cards. Throughout the study, animals are allowedaccess to food and water ad libitum.

Test compounds are administered via inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers are arranged so that an aerosol is simultaneouslydelivered to 6 individual chambers from a central manifold. Guinea pigsare exposed to an aerosol of a test compound or vehicle (WFI). Theseaerosols are generated from aqueous solutions using an LC Star NebulizerSet (Model 22F51, PARI Respiratory Equipment, Inc. Midlothian, Va.)driven by a mixture of gases (CO₂=5%, O₂=21% and N₂=74%) at a pressureof 22 psi. The gas flow through the nebulizer at this operating pressureis approximately 3 L/minute. The generated aerosols are driven into thechambers by positive pressure. No dilution air is used during thedelivery of aerosolized solutions. During the 10 minute nebulization,approximately 1.8 mL of solution is nebulized. This value is measuredgravimetrically by comparing pre- and post-nebulization weights of thefilled nebulizer.

The bronchoprotective effects of test compounds administered viainhalation are evaluated using whole body plethysmography at 1.5, 24, 48and 72 hours post-dose.

Forty-five minutes prior to the start of the pulmonary evaluation, eachguinea pig is anesthetized with an intramuscular injection of ketamine(43.75 mg/kg), xylazine (3.50 mg/kg) and acepromazine (1.05 mg/kg).After the surgical site is shaved and cleaned with 70% alcohol, a 2-3 cmmidline incision of the ventral aspect of the neck is made. Then, thejugular vein is isolated and cannulated with a saline-filledpolyethylene catheter (PE-50, Becton Dickinson, Sparks, Md.) to allowfor intravenous infusions of acetylcholine (Ach) or histamine in saline.The trachea is then dissected free and cannulated with a 14 G teflontube (#NE-014, Small Parts, Miami Lakes, Fla.). If required, anesthesiais maintained by additional intramuscular injections of theaforementioned anesthetic mixture. The depth of anesthesia is monitoredand adjusted if the animal responds to pinching of its paw or if therespiration rate is greater than 100 breaths/minute.

Once the cannulations are completed, the animal is placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula (PE-160, Becton Dickinson, Sparks, Md.) isinserted to measure pulmonary driving pressure (pressure). The teflontracheal tube is attached to the opening of the plethysmograph to allowthe guinea pig to breathe room air from outside the chamber. The chamberis then sealed. A heating lamp is used to maintain body temperature andthe guinea pig's lungs are inflated 3 times with 4 mL of air using a 10mL calibration syringe (#5520 Series, Hans Rudolph, Kansas City, Mo.) toensure that the lower airways do not collapse and that the animal doesnot suffer from hyperventilation.

Once it is determined that baseline values are within the range of0.3-0.9 mL/cm H₂O for compliance and within the range of 0.1-0.199 cmH₂O/mL per second for resistance, the pulmonary evaluation is initiated.A Buxco pulmonary measurement computer program enabled the collectionand derivation of pulmonary values.

Starting this program initiated the experimental protocol and datacollection. The changes in volume over time that occur within theplethysmograph with each breath are measured via a Buxco pressuretransducer. By integrating this signal over time, a measurement of flowis calculated for each breath. This signal, together with the pulmonarydriving pressure changes, which are collected using a Sensym pressuretransducer (#TRD4100), is connected via a Buxco (MAX 2270) preamplifierto a data collection interface (#'s SFT3400 and SFT3813). All otherpulmonary parameters are derived from these two inputs.

Baseline values are collected for 5 minutes, after which time the guineapigs are challenged with Ach or histamine. When evaluating themuscarinic antagonist effects, propanolol (5 mg/Kg, iv) (Sigma-Aldrich,St. Louis, Mo.) is administered 15 minutes prior to challenge with Ach.Ach (Sigma-Aldrich, St. Louis, Mo.) (0.1 mg/mL) is infused intravenouslyfor 1 minute from a syringe pump (s210iw, World Precision Instruments,Inc., Sarasota, Fla.) at the following doses and prescribed times fromthe start of the experiment: 1.9 μg/minute at 5 minutes, 3.8 μg/minuteat 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at 20minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes.Alternatively, bronchoprotection of test compounds is assessed in theacetylcholine challenge model without pretreatment with a beta blockingcompound.

When evaluating the β₂ adrenergic receptor agonist effects of testcompounds, histamine (25 μg/mL) (Sigma-Aldrich, St. Louis, Mo.) isinfused intravenously for 1 minute from a syringe pump at the followingdoses and prescribed times from the start of the experiment: 0.5μg/minute at 5 minutes, 0.9 μg/minute at 10 minutes, 1.9 μg/minute at 15minutes, 3.8 μg/minute at 20 minutes, 7.5 μg/minute at 25 minutes and 15μg/minute at 30 minutes. If resistance or compliance does not returnedto baseline values at 3 minutes following each Ach or histamine dose,the guinea pig's lungs are inflated 3 times with 4 mL of air from a 10mL calibration syringe. Recorded pulmonary parameters includerespiration frequency (breaths/minute), compliance (mL/cm H₂O) andpulmonary resistance (cm H₂O/mL per second). Once the pulmonary functionmeasurements are completed at minute 35 of this protocol, the guinea pigis removed from the plethysmograph and euthanized by carbon dioxideasphyxiation.

The data are evaluated in one of two ways:

(a) Pulmonary resistance (R_(L), cm H₂O/mL per second) is calculatedfrom the ratio of “change in pressure” to “the change in flow.” TheR_(L) response to ACh (60 μg/min, IH) is computed for the vehicle andthe test compound groups. The mean ACh response in vehicle-treatedanimals, at each pre-treatment time, is calculated and used to compute %inhibition of ACh response, at the corresponding pre-treatment time, ateach test compound dose. Inhibition dose-response curves for ‘R_(L)’ arefitted with a four parameter logistic equation using GraphPad Prism,version 3.00 for Windows (GraphPad Software, San Diego, Calif.) toestimate bronchoprotective ID₅₀ (dose required to inhibit the ACh (60μg/min) bronchoconstrictor response by 50%). The equation used is asfollows:Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hillslope)))where X is the logarithm of dose, Y is the response (% Inhibition of AChinduced increase in R_(L)). Y starts at Min and approachesasymptotically to Max with a sigmoidal shape.

(b) The quantity PD₂, which is defined as the amount of Ach or histamineneeded to cause a doubling of the baseline pulmonary resistance, iscalculated using the pulmonary resistance values derived from the flowand the pressure over a range of Ach or histamine challenges using thefollowing equation (derived from the equation used to calculate PC₂₀values (see Am. Thoracic Soc, 2000):

${PD}_{2} = {{antilog}\lbrack {{\log\mspace{14mu} C_{1}} + \frac{( {{\log\mspace{14mu} C_{2\;}} - {\log\mspace{14mu} C_{1}}} )( {{2\; R_{0}} - R_{1}} )}{R_{2} - R_{1}}} \rbrack}$

where:

-   -   C₁=concentration of Ach or histamine preceding C₂    -   C₂=concentration of Ach or histamine resulting in at least a        2-fold increase in pulmonary resistance (R_(L))    -   R₀=Baseline R_(L) value    -   R₁=R_(L) value after C₁    -   R₂=R_(L) value after C₂

Statistical analysis of the data is performed using a twotailed-Students t-test. A P-value <0.05 is considered significant.

Exemplified compounds of this invention are expected to produce adose-dependent bronchoprotective effect against MCh-inducedbronchoconstriction and His-induced bronchoconstriction. Generally, testcompounds having a potency (ID₅₀ at 1.5 h post-dose) of less than about300 μg/mL for ACh-induced bronchoconstriction and less than about 300μg/mL for His-induced bronchoconstriction in this assay are generallypreferred. Additionally, test compounds having a duration (PD T_(1/2))of brochoprotective activity of at least about 24 hours in this assayare generally preferred.

Assay Test Procedure G

Einthoven Model for Measuring Changes in Ventilation in Guinea Pigs

The bronchodilator activity of test compounds is evaluated in ananesthetized guinea pig model (the Einthoven model), which usesventilation pressure as a surrogate measure of airway resistance. See,for example, Einthoven (1892) Pfugers Arch. 51: 367-445; and Mohammed etal. (2000) Pulm Pharmacol Ther. 13(6):287-92. In this model, muscarinicantagonist and β₂ agonist activity is assessed by determining theprotective effects against methacholine (MCh) and histamine(His)-induced bronchoconstriction.

This assay is conducted using Duncan-Hartley guinea pigs (Harlan,Indianapolis, Ind.), weighing between 300 and 400 g.

The test compound or vehicle (i.e., sterile water) is dosed byinhalation (IH) over a 10 minute time period in a whole body exposuredosing chamber (R+S Molds, San Carlos, Calif.) using 5 mL of dosingsolution. Animals are exposed to an aerosol, which is generated from anLC Star Nebulizer Set (Model 22F51, PARI Respiratory Equipment, Inc.Midlothian, Va.) driven by Bioblend a mixture of gasses (5% CO₂; 21% O₂;and 74% N₂) at a pressure of 22 psi. Pulmonary function is evaluated atvarious time-points after inhalation dosing.

Forty five minutes prior to the start of pulmonary function evaluation,the guinea pigs are anesthetized with an intramuscular (IM) injection ofa mixture of ketamine (13.7 mg/kg/xylazine (3.5 mg/kg)/acepromazine(1.05 mg/kg). A supplemental dose of this mixture (50% of initial dose)is administered as needed. The jugular vein and carotid artery areisolated and cannulated with saline-filled polyethylene catheters(micro-renathane and PE-50, respectively, Beckton Dickinson, Sparks,Md.). The carotid artery is connected to a pressure transducer to allowthe measurement of blood pressure and the jugular vein cannula is usedfor IV injection of either MCh or His. The trachea is then dissectedfree and cannulated with a 14G needle (#NE-014, Small Parts, MiamiLakes, Fla.). Once the cannulations are complete, the guinea pigs areventilated using a respirator (Model 683, Harvard Apparatus, Inc., MA)set at a stroke volume of 1 mL/100 g body weight but not exceeding 2.5mL volume, and at a rate of 100 strokes per minute. Ventilation pressure(VP) is measured in the tracheal cannula using a Biopac transducer thatis connected to a Biopac (TSD 137C) pre-amplifier. Body temperature ismaintained at 37° C. using a heating pad. Prior to initiating datacollection, pentobarbital (25 mg/kg) is administered intraperitoneally(IP) to suppress spontaneous breathing and obtain a stable baseline. Thechanges in VP are recorded on a Biopac Windows data collectioninterface. Baseline values are collected for at least 5 minutes, afterwhich time guinea pigs are challenged IV non-cumulatively with 2-foldincremental doses of the bronchoconstrictor (MCh or His). When MCh isused as the bronchoconstrictor agent, animals are pre-treated withpropranolol (5 mg/kg, IV) to isolate the antimuscarinic effects of thetest compound. Changes in VP are recorded using the Acknowledge DataCollection Software (Santa Barbara, Calif.). After the completion ofstudy, the animals are euthanized.

Change in VP is measured in cm of water. Change in VP (cm H₂O)=peakpressure (after bronchoconstrictor challenge)−peak baseline pressure.The dose-response curve to MCh or His is fitted to a four parameterlogistic equation using GraphPad Prism, version 3.00 for Windows(GraphPad Software, San Diego, Calif.) The equation used is as follows:Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hillslope)))where X is the logarithm of dose, Y is the response. Y starts at Min andapproaches asymptotically to Max with a sigmoidal shape.

The percent inhibition of the bronchoconstrictor response to asubmaximal dose of MCh or His is calculated at each dose of the testcompound using the following equation: % Inhibition ofresponse=100−((peak pressure (after bronchoconstrictor challenge,treated)−peak baseline pressure (treated)*100%/(peak pressure (afterbronchoconstrictor challenge, water)−peak baseline pressure (water)).Inhibition curves are fitted using the four parameter logistic equationfrom GraphPad software. ID₅₀ (dose required to produce 50% inhibition ofthe bronchoconstrictor response) and Emax (maximal inhibition) are alsoestimated wherever appropriate.

The magnitude of bronchoprotection at different time-points afterinhalation of the test compound is used to estimate the pharmacodynamichalf-life (PD T_(1/2)). PD T_(1/2) is determined using a non-linearregression fit using a one-phase exponential decay equation (GraphPadPrism, Version 4.00): Y=Span*exp(−K*X)+Plateau; Starts at Span+Plateauand decays to Plateau with a rate constant K. The PD T_(1/2)=0.69/K.Plateau is constrained to 0.

Exemplified compounds of this invention are expected to produce adose-dependent bronchoprotective effect against MCh-inducedbronchoconstriction and His-induced bronchoconstriction. Generally, testcompounds having an ID₅₀ less than about 300 μg/mL for MCh-inducedbronchoconstriction and an ID₅₀ less than about 300 μg/mL forHis-induced bronchoconstriction at 1.5 hours post-dose in this assay arepreferred. Additionally, test compounds having a duration (PD T_(1/2))of bronchoprotective activity of at least about 24 hours in this assayare generally preferred.

Assay Test Procedure H

Inhalation Guinea Pig Salivation Assay

Guinea pigs (Charles River, Wilmington, Mass.) weighing 200-350 g areacclimated to the in-house guinea pig colony for at least 3 daysfollowing arrival. Test compound or vehicle are dosed via inhalation(IH) over a 10 minute time period in a pie shaped dosing chamber (R+SMolds, San Carlos, Calif.). Test solutions are dissolved in sterilewater and delivered using a nebulizer filled with 5.0 mL of dosingsolution. Guinea pigs are restrained in the inhalation chamber for 30minutes. During this time, guinea pigs are restricted to an area ofapproximately 110 sq. cm. This space is adequate for the animals to turnfreely, reposition themselves, and allow for grooming. Following 20minutes of acclimation, guinea pigs are exposed to an aerosol generatedfrom a LS Star Nebulizer Set (Model 22F51, PARI Respiratory Equipment,Inc. Midlothian, Va.) driven by house air at a pressure of 22 psi. Uponcompletion of nebulization, guinea pigs are evaluated at 1.5, 6, 12, 24,48, or 72 hrs after treatment.

Guinea pigs are anesthetized one hour before testing with anintramuscular (IM) injection of a mixture of ketamine 43.75 mg/kg,xylazine 3.5 mg/kg, and acepromazine 1.05 mg/kg at an 0.88 mL/kg volume.Animals are placed ventral side up on a heated (37° C.) blanket at a 20degree incline with their head in a downward slope. A 4-ply 2×2 inchgauze pad (Nu-Gauze General-use sponges, Johnson and Johnson, Arlington,Tex.) is inserted in the guinea pig's mouth. Five minutes later, themuscarinic agonist pilocarpine (3.0 mg/kg, s.c.) is administered and thegauze pad is immediately discarded and replaced by a new pre-weighedgauze pad. Saliva is collected for 10 minutes, at which point the gauzepad is weighed and the difference in weight recorded to determine theamount of accumulated saliva (in mg). The mean amount of salivacollected for animals receiving the vehicle and each dose of testcompound is calculated. The vehicle group mean is considered to be 100%salivation. Results are calculated using result means (n=3 or greater).Confidence intervals (95%) are calculated for each dose at each timepoint using two-way ANOVA. This model is a modified version of theprocedure described in Rechter, “Estimation of anticholinergic drugeffects in mice by antagonism against pilocarpine-induced salivation”Ata Pharmacol Toxicol, 1996, 24:243-254.

The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, is calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose. Theinhibition dose-response data are fitted to a four parameter logisticequation using GraphPad Prism, version 3.00 for Windows (GraphPadSoftware, San Diego, Calif.) to estimate anti-sialagogue ID₅₀ (doserequired to inhibit 50% of pilocarpine-evoked salivation). The equationused is as follows:Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hillslope)))where X is the logarithm of dose, Y is the response (% inhibition ofsalivation). Y starts at Min and approaches asymptotically to Max with asigmoidal shape.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ is usedto compute the apparent lung-selectivity index of the test compound.Generally, compounds having an apparent lung-selectivity index greaterthan about 5 are preferred.

Assay Test Procedure I

Radioligand Competition Binding Assay in Cloned Human Dopamine D2_(S)Receptor

In this assay, the binding affinity of test compounds for the humandopamine D2_(S) receptor in transfected CHO cells is determined using aradioligand binding assay (Grady et al., Proc. Natl. Acad. Sci. USA86:9762 (1989).

Cell membrane homogenates (5-10 μg protein) were incubated for 60minutes at 22° C. with 0.3 nM [³H]spiperone in the absence or presenceof a test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 120mM NaCl, 5 mM KCl, 5 mM MgCl₂ and 1 mM EDTA. The test compound was usedat a test concentration of 100 nM. Nonspecific binding was determined inthe presence of 10 μM (+)-butaclamol.

Following incubation, the samples were filtered rapidly under vacuumthrough glass fiber filters (GF/B, Packard) presoaked with 0.3%polyethyleneimine and rinsed several times with ice-cold 50 mM Tris-HClusing a 96-sample cell harvester (Unifilter, Packard). The filters weredried, and then counted for radioactivity in a scintillation counter(Topcount, Packard) using a scintillation cocktail (Microscint 0,Packard).

The results were expressed as a percent inhibition of the controlradioligand specific binding. Exemplified compounds of the inventiontested in this assay were found to have a percent inhibition of greaterthan about 30%, including greater than about 75%, at a concentration of100 nM. For example, the compound of Example 1 had a percent inhibitionof greater than about 75%; and the compound of Example 2 had a percentinhibition of greater than about 30%.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

1. A method of producing bronchodilation in a patient, the methodcomprising administering to a patient a bronchodilation-producing amountof a compound of formula I:

wherein one of G¹ and G² represents NH and the other represents S, NH, Oor CH₂; W represents O or NW; where W^(a) is hydrogen or (1-4C)alkyl;each R¹ is independently selected from (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(1a), —C(O)OR^(1b),—SR^(1c), —S(O)R^(1d), —S(O)₂R^(1e) and —NR^(1f)R¹g; where each ofR^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f) and R^(1g) isindependently hydrogen, (1-4C)alkyl or phenyl-(1-4C)alkyl; each R² isindependently selected from (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl,(3-6C)cycloalkyl, cyano, halo, —OR^(2a), —C(O)OR^(2b), —SR^(2c),—S(O)R^(2d), —S(O)₂R^(2e) and —NR^(2f)R^(2g); where each of R^(2a),R^(2b), R^(2c), R^(2d), R^(2e), R^(2f) and R^(2g) is independentlyhydrogen, (1-4C)alkyl or phenyl-(1-4C)alkyl; each R³ is independentlyselected from (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl,(3-6C)cycloalkyl, cyano, halo, —OR^(3a), —C(O)OR^(3b), —SR^(3c),—S(O)R^(3d), —S(O)₂R^(3e) and —NR^(3f)R^(3g); or two R³ groups arejoined to form (1-3C)alkylene, (2-3C)alkenylene or oxiran-2,3-diy1;where each of R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f) and R^(3g)is independently hydrogen or (1-4C)alkyl; R⁴ represents a divalenthydrocarbon group containing from 4 to 28 carbon atoms and optionallycontaining from 1 to 10 heteroatoms selected independently from halo,oxygen, nitrogen and sulfur, provided that the number of contiguousatoms in the shortest chain between the two nitrogen atoms to which R⁴is attached is in the range of from 4 to 16; R⁵ represents hydrogen or(1-4C)alkyl; R⁶ represents hydrogen or hydroxyl; each R^(7a) and R^(7b)is independently selected from hydrogen, (1-4C)alkyl, hydroxy andfluoro; a is 0 or an integer of from 1 to 3; b is 0 or an integer offrom 1 to 3; c is 0 or an integer of from 1 to 4; d is 0 or an integerof from 1 to 5; and m is 0 or an integer of from 1 to 3; or apharmaceutically acceptable salt or stereoisomer thereof
 2. A method oftreating chronic obstructive pulmonary disease or asthma, the methodcomprising administering to a patient in need of treatment a compound offormula I:

wherein one of G¹ and G² represents NH and the other represents S, NH, Oor CH₂; W represents O or NW; where W^(a) is hydrogen or (1-4C)alkyl;each R¹ is independently selected from (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(1a), —C(O)OR^(1b),—SR^(1c), —S(O)R^(1d), —S(O)₂R^(1e) and —NR^(1f)R¹g; where each ofR^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f) and R^(1g) isindependently hydrogen, (1-4C)alkyl or phenyl-(1-4C)alkyl; each R² isindependently selected from (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl,(3-6C)cycloalkyl, cyano, halo, —OR^(2a), —C(O)OR^(2b), —SR^(2c),—S(O)R^(2d), —S(O)₂R^(2e) and —NR^(2f)R^(2g); where each of R^(2a),R^(2b), R^(2c), R^(2d), R^(2e), R^(2f) and R^(2g)is independentlyhydrogen, (1-4C)alkyl or phenyl-(1-4C)alkyl; each R³ is independentlyselected from (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl,(3-6C)cycloalkyl, cyano, halo, —OR^(3a), —C(O)OR^(3b), —SR^(3c),—S(O)R^(3d), —S(O)₂R^(3e) and —NR^(3f)R³g; or two R³ groups are joinedto form (1-3C)alkylene, (2-3C)alkenylene or oxiran-2,3-diy1; where eachof R^(3a), R^(3b), R^(3c), R^(3d), R^(3e), R^(3f) and R^(3g) isindependently hydrogen or (1-4C)alkyl; R⁴ represents a divalenthydrocarbon group containing from 4 to 28 carbon atoms and optionallycontaining from 1 to 10 heteroatoms selected independently from halo,oxygen, nitrogen and sulfur, provided that the number of contiguousatoms in the shortest chain between the two nitrogen atoms to which R⁴is attached is in the range of from 4 to 16; R⁵ represents hydrogen or(1-4C)alkyl; R⁶ represents hydrogen or hydroxyl; each R^(7a) and R^(7b)is independently selected from hydrogen, (1-4C)alkyl, hydroxy andfluoro; a is 0 or an integer of from 1 to 3; b is 0 or an integer offrom 1 to 3; c is 0 or an integer of from 1 to 4; d is 0 or an integerof from 1 to 5; and m is 0 or an integer of from 1 to 3; or apharmaceutically acceptable salt or stereoisomer thereof.
 3. The methodof claim 1, wherein a dose of the compound for providing bronchodilationranges from about 10 μg/day to about 200 μg/day.
 4. The method of claim2, wherein the compound is administered by inhalation.
 5. The method ofclaim 2, wherein the compound is administered in multiple doses per day.6. The method of claim 2, wherein the compound is administered in asingle daily dose.
 7. The method of claim 2, wherein a dose of thecompound for treating chronic obstructive pulmonary disease or asthmaranges from about 10 μg/day to about 200 μg/day.
 8. The method of claim2, wherein R⁴ is a divalent group of the formula:—(R^(4a))_(d)-(A¹)_(e)-(R^(4b))_(f)-Q-(R^(4c))_(g)-(A²)_(h)-(R^(4d))_(i)—wherein d, e, f, g, h and i are each independently selected from 0 and1; R^(4a), R^(4b), R^(4c) and R^(4d) are each independently selectedfrom (1-10C)alkylene, (2-10C)alkenylene and (2-10C)alkynylene, whereineach alkylene, alkenylene or alkynylene group is unsubstituted orsubstituted with from 1 to 5 substituents independently selected from(1-4C)alkyl, fluoro, hydroxy, phenyl and phenyl-(1-4C)alkyl; A¹ and A²are each independently selected from (3-7C)cycloalkylene,(6-10C)arylene, —O-(6- 10C)arylene, (6- 10C)arylene-O—,(2-9C)heteroarylene, —O—(2-9C)heteroarylene, (2-9C)heteroarylene-O— and(3-6C)heterocyclene, wherein each cycloalkylene is unsubstituted orsubstituted with from 1 to 4 substitutents selected independently from(1-4C)alkyl, and each arylene, heteroarylene or heterocyclene group isunsubstituted or substituted with from 1 to 4 substituents independentlyselected from halo, (1-4C)alkyl, (1-4C)alkoxy, —S-(1-4C)alkyl,—S(O)-(1-4C)alkyl, —S(O)₂-(1-4C)alkyl, —C(O)O(1-4C)alkyl, carboxy,cyano, hydroxy, nitro, trifluoromethyl and trifluoromethoxy; Q isselected from a bond, —O—, —C(O)O—, —OC(O)—, —S—, —S(O)—, —S(O)₂—,—N(Q^(a))C(O)—, —C(O)N(Q^(b))-, —N(Q^(c))S(O)₂—, —S(O)₂N(Q^(d))-,—N(Q^(e))C(O)N(Q^(f)) -, —N(Q^(g))S(O)₂N(Q^(h))-, —OC(O)N(Q^(i))-,—N(Q^(j))C(O)O— and —N(Q^(k)); Q_(a), Q^(b), Q^(C), Q^(d), Q^(e), Q^(f),Q^(g), Q^(h) , Q^(i), Q^(j) and Q^(k) are each independently selectedfrom hydrogen, (1-6C)alkyl, A³ and (1-4C)alkylene-A⁴, wherein the alkylgroup is unsubstituted or substituted with from 1 to 3 substituentsindependently selected from fluoro, hydroxy and (1-4C)alkoxy; ortogether with the nitrogen atom and the group R^(4b) or R^(4c) to whichthey are attached, form a 4-6 membered azacycloalkylene group; A³ and A⁴are each independently selected from (3-6C)cycloalkyl, (6-10C)aryl,(2-9C)heteroaryl and (3-6C)heterocyclyl, wherein each cycloalkyl isunsubstituted or substituted with from 1 to 4 substitutents selectedindependently from (1-4C)alkyl and each aryl, heteroaryl or heterocyclylgroup is unsubstituted or substituted with from 1 to 4 substituentsindependently selected from halo, (1-4C)alkyl and (1-4C)alkoxy.
 9. Themethod of claim 8, wherein R⁴ is a divalent group of the formula:—(R^(4a))_(d)— where R^(4a) is (4-10C)alkylene.
 10. The method of claim8, wherein R⁴ is —(CH₂)₄—, —(CH₂)₅—, —(CH₂)₆—, —(CH₂)₇—, —(CH₂)₈—,—(CH₂)₉, or —(CH₂)₁₀—.
 11. The method of claim 8, wherein R⁴ is adivalent group of the formula:—(R^(4a))_(d)-(A²)_(h)-(R^(4d))_(i)— where R^(4a) is (1-10C)alkylene; A²is (6-10C)arylene or (2-9C)heteroarylene; and R^(4d) is (1-10C)alkylene.12. The method of claim 8, wherein R⁴ is a divalent group of theformula:—(R^(4a))_(d)-Q-(A²)_(h)-(R^(4d))_(i)— where Q is —O— or —N(Q^(k))-;Q^(k) is hydrogen or (1-3C)alkyl; R^(4a) is (1-10C)alkylene; A² is(6-10C)arylene or (2-9C)heteroarylene; and R^(4d) is (1-10C)alkylene.13. The method of claim 8, wherein R⁴ is a divalent group of theformula:—(R^(4a))_(d)-(A¹)_(e)-(R^(4b))_(f)-Q-(R^(4c))_(g)-(A²)_(h)-(R^(4d))_(i)— where Q is—N(Q^(a))C(O)— or —C(O)N(Q^(b))-.
 14. The method of claim 2, wherein thecompound is a compound of formula Ia:

or a pharmaceutically acceptable salt or stereoisomer thereof.
 15. Themethod of claim 2, wherein the compound is a compound of formula Ib:

or a pharmaceutically acceptable salt or stereoisomer thereof.
 16. Themethod of claim 2, wherein the compound is a compound of formula Ic:

or a pharmaceutically acceptable salt or stereoisomer thereof.
 17. Themethod of claim 2, wherein the compound is a compound of formula Id:

or a pharmaceutically acceptable salt or stereoisomer thereof.